Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT The treatment of metastatic clear cell renal cell carcinoma (ccRCC), the most common and lethal form of renal cell carcinoma, has been revolutionized by therapies directed at vascular endothelial growth factor (VEGF) and immune checkpoint blockade (ICB) therapies. Despite this progress, the majority of patients with advanced disease will develop treatment resistance and ultimately succumb to their disease, making alternative therapeutic strategies a critical need. We have previously demonstrated that ccRCC is highly immune infiltrated, mostly by T cells and antigen-presenting cells (APCs), in particular, tumor-associated macrophages (TAMs). We and others have further demonstrated that TAMs are associated with resistance to both ICB and VEGF-directed therapy, a process thought to be mediated, in part, by T cell exhaustion. However, TAMs are phenotypically and functionally diverse, and our preliminary evidence suggests that only some TAM subpopulations are associated with therapeutic resistance. We hypothesize that specific TAM subpopulations can impact tumor control and response to therapy by presenting antigen to and influencing T cell phenotype, and therefore that specific inhibition of these TAMs may increase response to the therapies. To test these ideas, we propose first to finely characterize APC populations in 3 unique sets of human tumors: treatment-naïve, responsive to combination therapy, and resistant to the therapy. We then assess APC populations as predictors of patient outcome to construct new predictive models. Next, to uncover how antigen presentation by TAMs locally modulates anti-tumor T cell responses, we will use a novel genetically faithful, immunocompetent murine model. Specifically, we will assess immune cell phenotypes and tumor growth in mosaic mice with TAM-specific genetic ablation of antigen presentation function, as well as assessing TAMs in vitro for ability to present antigen and influence T cell states. Finally, we will take advantage of indications that various macrophage-directed drugs in clinical development may selectively inhibit different TAM subsets. After determining which TAM subpopulations are associated to resistance to current therapies for ccRCC (anti-PD-1, anti-PD-L1, and the VEGF receptor inhibitor cabozantinib), we will use the mouse model to assess whether resistance can be overcome by treating with a macrophage-directed drug that inhibits the resistance-associated TAM subsets. In summary, the proposed research will yield a detailed atlas of APC and T cell states in ccRCC, a novel model to predict patient outcome, and an understanding of the role of antigen presentation by TAMs in ccRCC. Further, the proposed research may reveal a means of overcoming resistance to widely used therapies for ccRCC. Therefore, this work can open the door for precision-based TAM inhibition strategies to overcome treatment resistance in ccRCC and other immune infiltrated tumors.

NIH Research Projects · FY 2025 · 2021-04

Project Abstract Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft tissue sarcomas that occur in distinct clinical settings: neurofibromatosis 1 (NF1)-associated (45%), sporadic (45%) or radiotherapy (RT)-associated (10%). MPNSTs metastasize early and are resistant to radiotherapy and systemic chemotherapy and have poor prognosis. Irrespective of the clinical settings, MPNSTs share the same molecular pathway inactivation in NF1 (>90%, hence NF1-deficient), Polycomb repressive complex 2 (PRC2), and CDKN2A through biallelic genetic alterations, suggesting that they can be molecularly targeted similarly. NF1- deficient plexiform neurofibroma responds well to MEK inhibitor (MEKi) treatment clinically; however, NF1- deficeint MPNSTs arising from plexiform neurofibromas are universally resistant to MEKi, suggesting intrinsic resistance in the more aggressive form of peripheral nerve sheath tumors. Using MPNST patient tumor samples and preclinical MPNST models, we have uncovered that PRC2-loss leads to PDGRA upregulation; MEKi treatment resulted in feedback upregulation of PDGFRB irrespective of the PRC2 status. The convergence of the PDGFR pathway activation by different mechanisms points to a novel therapeutic opportunity to target the PDGFR pathway to overcome MEKi resistance in MPNST. Combination of a novel PDGFRA/B inhibitor, ripretinib with a MEKi leads to synergistic growth inhibition of MPNST in vitro and in vivo. We hypothesize that PDGFRA/B pathway activation represent a central resistance mechanism to MEKi and combined targeting of the PDGFR and MAPK pathways with ripretinib (pan-PDGFRA/B inhibitor) and binimetinib (MEKi) may present an effective therapeutic strategy in NF1-deficient MPNST. Here, we propose to investigate the tumor heterogeneity and cellular plasticity involved in tumor evolution and adaptive resistance to binimetinib and combination of ripretinib and binimetinib, using well-defined preclinical MPNST in vitro and in vivo model systems and single-cell analysis including single cell RNA-seq (scRNA-seq) and a novel barcoding system. Additionally, we propose a collaborative clinical investigation between CCR/NCI (Drs. Widemann/Shern) and MSKCC (Dr. Chi) to conduct a proof-of-concept phase I/II study of the combination of ripretinib and binimetinib in patients with NF1-deficient MPNST. In this trial, we will assess and optimize the evaluation of MAPK pathway inhibition to the ripretinib/binimetinib combination therapy using traditional ERK phosphorylation and newly established custom Pea3-family ETS-regulated MAPK signature. Further, we will also investigate the tumor heterogeneity and cellular plasticity in tumor evolution and resistance mechanisms to the ripretinib/binimetinib combination using targeted NGS, scRNA-seq and integrative analysis. The proposal leverages the synergistic expertise and resources at MSKCC and CCR/NCI. We believe that these studies will generate mechanistic insight of therapeutic resistance and provide the pivotal clinical and translational information for future definitive trials, with the potential to change the clinical practice in MPNST.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Parkinson's disease (PD) is a movement disorder that involves the selective loss of midbrain dopamine (mDA) neurons in the substantia nigra. Human stem cells, such as embryonic (hESCs) and induced pluripotent (hiPSCs), represent a powerful technology to study and potentially treat PD. Methods to generate mDA neurons from human stem cells have been pioneered by our group. Such work enabled applications of mDA neurons for modeling PD in a dish and for the development of cell-based therapies. In fact, based on our work, the transplantation of human mDA neurons is at the verge of clinical testing in PD. Despite such progress, current strategies for generating mDA neurons are suboptimal and the resulting cells do not match all the molecular features of mDA neurons in the brain. In addition, there are no reliable purification methods to specifically enrich for mDA neurons. The lack of such methods is a problem, particularly in disease modeling, where mDA neurons are compared across cell lines from many PD patients and where variability in yield can be a major confounding factor. Furthermore, the use of purified mDA neurons will allow more precise transplantation studies to define optimal graft composition. Another important challenge is the limited survival of mDA neurons after transplantation (~10% of grafted cells), a problem that remains unresolved, and that can cause variability in cell dosing and complicate the routine application of this technology. A final challenge is the lack of knowledge how to preferentially generate mDA neurons of either A9 (substantia nigra) or A10 (ventral tegmental area) identity. Both A9 and A10 are mDA neurons, but they represent subtypes with different molecular and functional properties, and with A9 being the desired subtype for disease modeling and cell therapy in PD. Here, we propose three specific aims to address these outstanding questions. In Aim1, based on exciting preliminary data, we will refine our mDA neuron differentiation strategy to obtain mDA neurons with improved molecular and functional properties and a sorting method that will enable routine purification of mDA neurons. We propose the use of single cell gene expression analysis to assess whether mDA neurons under such improved conditions more fully match mDA neurons in the developing or adult brain. In Aim 2, we will define the factors that limit survival of mDA neurons upon cell transplantation. We have developed a very promising, CRISPR-based screening technology to define survival factors, and already identified candidates acting either directly within mDA neurons or via the host environment. Finally, in Aim 3, we will use single cell gene expression and chromatin accessibility studies to map A9/A10 subtype diversity of mDA neurons from human stem cells. The results from those in-depth single cell profiling studies will be used to identify and test factors that are functionally important in subtype specification. Each of the three aims addresses a critical and complementary challenge in the mDA field towards unlocking the full potential of human stem cell-derived mDA neurons for cell therapy and human disease modeling.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract: As many as 35% of older adult cancer survivors (OACS; ≥65 years, ≥1-year post- treatment) have clinically significant depression. Presently, 64% of cancer survivors are ≥65 years old, and that number will increase to 73% by the year 2040. Thus, the number of depressed OACS will exceed 6 million as the U.S. population continues to age. Even if OACS pursue depression treatment, they may not receive developmentally appropriate services because the geriatric mental health workforce is too small to meet the needs of this growing population in oncology. Therefore, OACS are a uniquely high-risk group for untreated depression in need of novel, widely disseminable treatment strategies. Yet, no studies have evaluated the implementation of evidence-baseddepression treatment for older adults through cancersurvivorship clinics. The current proposal adopts an implementation science framework to target depression in OACS. Dr. Saracino proposes the adaptation of Behavioral Activation (BA) as a promising, straightforward treatment to increase OACS’ engagement in evidence-based depression treatment. Using an exploratory sequential mixed methods design, including a small open pilot and randomized control trial, this study has three specific aims: 1) To obtain feedback from key stakeholders (i.e., OACS, social workers, survivorship clinic clinicians ) to revise the BA manual content and procedures and develop an understanding of barriers to treatment engagement in OACS; 2) To evaluate implementation outcomes (i.e., appropriateness, acceptability, adoption, feasibility, fidelity, penetration, and sustainability) of BA in cancer survivorship; and 3) To determine the preliminary effects of BA on depression (primary outcome), anxiety, coping, and behavioral activation (secondary outcomes) compared to a Supportive Psychotherapy (SP) control arm. Completion of this project will facilitate Dr. Saracino’s long term career goal of becoming an independent investigator with expertise in the dissemination and implementation (D&I) of mental health screening and treatment for older adults across the cancer trajectory . In the context of a supportive, enriching environment with a strong mentorship team of national and inte rnational experts, Dr. Saracino will acquire advanced training in 5 areas critical to her success as an independent investigator: 1) Cancer Survivorship; 2) Geriatric Mental Health Research; 3) D&I Science; 4) Mixed Methods; and 5) Longitudinal Data Analysis. Dr. Saracino has outlined a comprehensive 5-year training plan that includes guided mentoring, formal coursework, seminars, onlinetrainings, national conferences, and hands-on researchactivities that directly parallel her training goals. This proposal is responsive to the NCI’s call for research on cancer survivorship and older adults, and the Cancer Moonshot Initiative Blue Ribbon Panel Working Group on Implementation Science’s recommendation that implementation research should be conducted on how to tailor and deploy evidence-based interventions at multiple levels (e.g. individuals, providers, systems, communities) and in different clinical and community settings to maximize symptom control.

NIH Research Projects · FY 2025 · 2021-04

Selpercatinib (LOXO-292) is a highly active selective RET inhibitor explored on an ongoing registrational program (LIBRETTO-001 phase 1/2 trial) in RET-dependent cancers (US FDA approval in 2020 for RET fusion-positive lung/thyroid cancer and RET-mutant thyroid cancer). Unfortunately, resistance is uncharacterized and remains a liability. This proposal anticipates and addresses this unmet need by identifying and functionally characterizing mechanisms of intrinsic and acquired genomic and non-genomic resistance to selective RET inhibition in RET-dependent cancers. To accomplish this, we will leverage unique clinical, computational, and translational resources at our disposal. Aim 1 will identify determinants of intrinsic resistance to RET inhibition in RET-dependent cancers. Pre-treatment biopsies of selpercatinib responders and non-responders will undergo targeted/whole exome sequencing. Computational analysis will explore the role of clonality, allelic imbalance, and co-mutational signatures relative to selpercatinib response and progression-free survival. Aim 2 will establish the mechanisms of acquired resistance to selective RET inhibition. Paired pre-treatment and post-progression tumor biopsies and longitudinal cell-free (cf)DNA (baseline, on-treatment, at/post- progression) from LIBRETTO-001 patients will be profiled. Utilizing paired samples will allow for the identification of emergent genomic and non-genomic (including histologic/EMT transformation) resistance mechanisms. In addition, plasma profiling will allow for a dynamic assessment of selpercatinib resistance that captures the consequences of serial genomic evolution. Aim 3 will functionally characterize resistance to selective RET inhibition. A unique and rich library of patient-derived models of treatment-naïve and RET inhibitor resistant RET-dependent cancers will be augmented by ongoing prospective collection and model development from LIBRETTO-001 and commercial use. In these models, on-target (secondary RET mutations) and off-target (MET/PI3K/KRAS/MDM2 activation) resistance mechanisms will be functionally characterized in terms of cell/tumor viability, receptor tyrosine kinase activation, and downstream signaling dependencies. Novel therapeutic strategies, specifically RET tyrosine kinase inhibitor type switching (on-target resistance) and combinatorial therapies (off-target resistance), will be explored in vitro and in vivo. Optimal combination therapies will then be employed in compassionate use programs to confirm their effectiveness and provide tailored, life-saving treatment to patients. In addition, patients with on-target resistance will be treated on-protocol (Drilon PI) with the next-generation RET inhibitor, TPX-0046. This proposal has both immediate and long-term relevance considering that about 400 patients have been treated with selpercatinib around the world on trial and the potential approval and rapid adoption of this drug by multiple regulatory agencies. These current and future patients are in dire need of strategies to re-establish durable disease control after progression on selective RET inhibition.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage that include Langerhans Cell Histiocytosis (LCH), Erdheim-Chester Disease (ECD), Juvenile Xanthogranuloma (JXG), and Rosai-Dorfman Disease (RDD). Although the pathogenesis of histiocytoses was previously obscure, it is now known that nearly every patient with these disorders has a mutation activating MAP kinase (MAPK) signaling, including BRAFV600E mutations in 50% of LCH and ECD cases. BRAF inhibition is efficacious for those patients with BRAFV600E-mutant disease and our clinical trial led to FDA approval of vemurafenib for BRAFV600E-mutant ECD in 2017. Correlative analyses from this study revealed that the allelic burden of mutant cell-free DNA in the plasma is a dynamic and reliable biomarker of therapeutic response in BRAFV600E-mutant histiocytosis. Prelim- inary data: More recently, a wide spectrum of genomic alterations in kinase signalling components were iden- tified in BRAFV600E-wildtype (WT) histiocytic neoplasms. Diverse mutations in MEK1/2 and ARAF are among the most common, accounting for >50% of BRAFV600 WT cases. Patients with BRAFV600-WT histiocytosis have also been treated with MEK inhibition, both within a phase 2 clinical trial (NCT02649972) and in clinical prac- tice. Knowledge gap: In contrast to the near-universal activity of BRAF inhibition for BRAFV600E-mutant histio- cytosis, patients with BRAFV600-WT histiocytosis exhibit heterogeneity in their responses to MEK inhibition, the basis of which remains unknown. Moreoever, many alterations in components of the MAPK pathway in histio- cytoses have not been functionally characterized. The hypothesis of this study is that tumor cell-intrinsic ge- netic alterations correlate with response to MEK inhibition in histiocytosis. This hypothesis will be tested by evaluating therapeutic responses to MEK inhibition in our cohort of BRAFV600-WT histiocytosis patients with diverse MAPK pathway mutations, and with innovative genomic analysis of plasma cell-free DNA to identify biomarkers of response. Memorial Sloan Kettering Cancer Center is the leading referral center in the U.S. for adults with histiocytosis—making it ideally suited to conduct this work. In parallel, the mechanistic and thera- peutic implications of mutations in MEK1/2 and ARAF (which represent the most commonly mutated genes in BRAFV600-WT patients) will be investigated. Although ARAF and MEK1/2 mutations are recurrent across many cancers, the unique enrichment of these mutations in histiocytoses provides an opportunity to functionally dis- sect mechanisms by which ARAF and MEK1/2 regulate MAPK signalling and drive cancer. Impact: This pro- ject will improve our understanding of histiocytosis pathogenesis, mechanisms of MAPK pathway activation, and determinants of response to MEK inhibition. Aim 1: Characterize the clinical and molecular response of histiocytosis to MEK inhibition in a prospectively treated patient cohort, including an ongoing phase 2 clinical trial. Aim 2. Understand the biochemical impact and therapeutic implications of MEK1/2 and ARAF mutations in patients with histiocytoses.

NIH Research Projects · FY 2025 · 2021-04

Patients with advanced thyroid cancer are frequently refractory to radioiodine (RAI) therapy. Oncoproteins that constitutively activate MAPK signaling suppress expression of genes that control thyroid differentiated function and response to RAI, which can be reversed, at least in part, with RAF or MEK inhibitors in mouse models and in pilot clinical trials. These treatments are less effective in BRAFV600E-mutant cancers, which we showed to be due to adaptive resistance to RAF or MEK inhibitors. Profound MAPK pathway blockade increases iodide uptake but does not increase iodine retention time in mouse BrafV600E PTCs. This is because these compounds relieve negative feedback inputs that increase PI3K signaling, which impairs expression of genes required for iodide oxidation and incorporation into thyroglobulin (TG). This can be rescued by combined treatment with a pan-PI3K inhibitor. The goal of this proposal is to build on the progress so far to attain greater efficacy of redifferentiation therapies in patients with thyroid cancer who are most likely to benefit. We propose to do this by: 1. Investigating whether selective PI3K isoform or HER kinase inhibitors increase iodide retention and RAI efficacy in the context of MAPK blockade. 2. Test the effects of the RAF inhibitor vemurafenib and the pan-PI3K inhibitor copanlisib on expression of iodide organification genes and how this relates to lesional 124I uptake and retention time in patients with RAI-refractory BRAFV600E metastatic thyroid cancer. 3. Identify molecular predictors of RAI efficacy in patients who had exceptional structural responses to conventional or MAPK-inhibitor enhanced RAI treatment using a case-control study design. 4) Develop novel therapeutic bispecific antibodies to redirect polyclonal T cells to target adaptive responses to MAPK inhibitors.

NIH Research Projects · FY 2025 · 2021-02

ABSTRACT The goal of the proposed project is to address how neuronal stress triggered by amyloid-beta and tau oligomeric species induces protein connectivity dysfunctions and alters protein-to-neuronal circuit-to-organ level function. Our focus in on synaptic dysfunction and cognitive deficits in Alzheimer's disease (AD). The hypothesis behind our investigation is that upon entry, the molecular stress triggered by amyloid-beta and tau oligomeric species induces a maladaptive rewiring in the connectivity, and in turn the function of large subsets of downstream neuronal proteins and their networks, through pathologic chaperome scaffolds termed epichaperomes. This hypothesis is supported by preliminary data obtained by the Chiosis lab showing that neuronal lineages are especially prone to form epichaperomes following stressors, and that most vulnerable to epichaperomes are protein pathways with key roles in synaptic plasticity. Additional preliminary experiments supporting feasibility of our experimental plan are provided by studies from the Arancio laboratory and others demonstrating that A and tau oligomers alter synaptic connectivity leading to memory loss. Our preliminary observation that dismantling the pathologic epichaperome structures into normal, folding chaperones rebalances protein network connectivity and functionality to those seen in physiological conditions, are also in support of our scientific premise. To execute these studies, we use iPSC-derived cellular models and mouse models of AD and combine the synergistic expertise of Drs. Arancio (synaptic plasticity, biology of AD), Chiosis (chemical biology of pathologic protein networks, translational research), Fraser (mouse models of AD and AD biology), Zhou (iPSC models in disease) and Mertens (consultant on hiPSC and iN-based cellular models for synaptic function study in AD). We expect that our studies will deliver proteome-wide functional insights and comprehensive, mechanistic understanding into how A and tau oligomers lead to synaptic failure and cognitive defects. In addition to providing new insights into AD biology, our studies have immediate translational applications. With an epichaperome therapeutic discovered by the Chiosis lab moving into Phase 2 clinical evaluation in AD, hypotheses tested within the present proposal may have immediate impact in human AD.

NIH Research Projects · FY 2025 · 2021-02

Project Summary Natural killer (NK) cells comprise an important arm of the host innate immune system that detects and eliminates virus-infected cells. Newborns and immune-compromised patients lacking NK cells are extremely susceptible to viral infection. In particular, human cytomegalovirus (HCMV) can cause severe health complications or be life-threatening in these individuals. Mouse cytomegalovirus (MCMV) is an accurate and robust model for investigating NK cell responses against HCMV. Using MCMV infection in mice, we have discovered that NK cells possess novel adaptive immune features such as clonal expansion and long-lived memory. In the past decade, our laboratory has uncovered many of the cellular and molecular mechanisms underlying NK cell memory. Our long-term goals are to understand the general biology of NK cells, and the molecular basis by which these powerful innate lymphocytes can mediate protection against pathogen invasion. To this end, we have recently identified several transcriptional and metabolic pathways that may influence the NK cell response against MCMV infection. Based on this exciting preliminary data, our current R01 grant proposes to use cutting edge metabolomics and newly engineered transgenic mouse models to study how metabolism in antiviral NK cells in transcriptionally regulated. In Aim 1, we seek to understand how proinflammatory cytokines and the STAT family of transcription factors control of NK cell metabolism during MCMV infection. In Aim 2, we will determine the requirement for aerobic glycolysis and fatty acid oxidation in antiviral NK cells using conditional ablation of genes encoding LDHA and CPT1a, respectively. In Aim 3, we will determine whether the transcription factor Bhlhe40 regulates mitochondrial metabolism and fitness in effector NK cells fighting MCMV infection. Altogether, the studies in this R01 proposal will greatly increase our understanding of the underlying transcriptional and metabolic mechanisms whereby NK cells contribute to host defense during viral infection, and establish novel translational paradigms for harnessing the NK cell compartment for immunization and therapeutic strategies against infectious diseases.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Patients with anaplastic thyroid cancer (ATC) have a median survival of 6 months. Chemotherapy in combination with surgery and radiation may modestly extend survival of ATC confined to the neck but offers minimal benefit in patients with metastatic disease. Recently, combination treatment using the RAF inhibitor dabrafenib with the MEK inhibitor trametinib showed a 69% ORR and dramatic tumor regression in BRAFV600E- driven ATCs, which is the first meaningful advance in the treatment of this disease. In contrast, response to this same combination in BRAF-driven differentiated thyroid cancer (DTC) is 33%. The remarkable dab/tram effects in BRAF-ATCs provide a roadmap to explore the therapeutic vulnerabilities in this disease. A hallmark of ATCs is their heavy infiltration with tumor-associated macrophages (TAMs) with M2 polarization. TAMs are associated with a worse prognosis in thyroid and other cancers, attributed in part to their suppression of immune surveillance. Although ATCs are also enriched for T cells, a pilot trial of immune checkpoint blockade (ICB) showed no efficacy in this disease, including in two ATCs with microsatellite-instability, predicted to harbor numerous neoantigens. Based on a mouse model of Braf-driven ATC that we developed, which recapitulates the immune milieu of the human disease, we propose that profound MAPK pathway inhibition blocks production of tumor-derived cytokines governing TAM recruitment and maintenance, leading to their depletion and the consequent de-repression of T cell cytotoxicity. We hypothesize that MAPK blockade primarily accounts for this TME response, and that TAMs and MAPK regulation of ATC antigen presentation play a central role in the process. To understand the relative contribution of these mechanisms we will: 1) Identify ATC cell-derived cytokines that recruit infiltrating myeloid cell populations. We identified a MAPK- driven cytokine panel in BRAF-ATCs and will determine individual cytokine contributions to myeloid recruitment using genetic and pharmacological approaches. 2) Determine the role of TAMs in response to therapy. We will first investigate whether TAMs block T-cell responses to model antigens (Pmel and/or Tyrp1) in ATCs in vivo. We will perform genetic or pharmacological depletion of TAMs to determine the optimal approach to enhance T cell responses, then determine the effects of TAM depletion or repolarization on T cell response to tumor cell autonomous neoantigens, and whether these can further enhance efficacy of ICB in mouse Braf/p53 ATCs. 3) Determine the role of tumor cell antigen presentation in T cell response following MAPK inhibition and whether the magnitude or duration of response can be enhanced by Pd1 blockade. 4) Determine sequential changes in the immune landscape of human BRAFV600E ATCs in response to preoperative treatment with dab/tram and during a combination trial of dab/tram with the PD1 inhibitor Cemiplimab.

NIH Research Projects · FY 2025 · 2021-01

Our data indicate that in select settings activation of acid sphingomyelinase (ASMase)/ceramide signaling in tumor endothelial cells by radiation and certain chemotherapies synergizes with direct tumor cell damage to impact outcome. ASMase is a lysosomal hydrolase preferentially expressed in endothelial cells up to 20-fold compared with other mammalian cells. Mechanistically, endothelial ASMase activation leads within min to formation of plasma membrane ceramide-rich platforms (CRPs), macrodomains that organize apoptotic signaling programs. Support for our concept derives from studies showing xenografts of all histologies, when implanted in asmase-/- host mice become resistant to gemcitabine, paclitaxel, etoposide, and high single dose radiotherapy, effects reversed by exclusive adenoviral asmase gene delivery to tumor microvasculature. We recently discovered VEGF is the principal inhibitor of endothelial ASMase, and that anti-angiogenic drugs de-repress ASMase, amplifying tumor responses to anti-cancer therapies, but only under specific conditions. We found irrespective of t1/2 or anti-angiogenic class, these drugs enhance endothelial apoptosis and tumor response only if scheduled at 1-2h preceding chemotherapy, as ASMase can be de-repressed for only 1-2h. Based on these data, the MSK Sarcoma Service performed a Phase II trial that showed sphingolipid-based timing of bevacizumab vs. conventional synchronous timing improved metastatic sarcoma response to the cytidine analogue gemcitabine from 38 to 93% (p=0.0024; Tap and Kolesnick, unpublished). The current application will help establish the mechanism by which temporal delivery of a short-acting anti-angiogenic drug simultaneously enhances gemcitabine-induced endothelial and tumor cell apoptosis. The overarching hypothesis of this application is that the principal nucleoside transporter in mammalian cells, ENT1, required for gemcitabine uptake, is not constitutively “on” as generally accepted but must insert into CRPs on endothelial and tumor cells for functionalization. This new membrane-based mechanism of gemcitabine action will be explored in 3 aims designed to examine mechanism of ENT1 functionalization via CRPs in both endothelial and sarcoma cells, VEGF inhibition of ASMase-generated CRPs, and pharmacologic strategies to enhance endothelial ASMase- ceramide signaling in vivo to improve ENT1-mediated gemcitabine uptake and cell death. A major concept to be explored is that gemcitabine-induced ASMase secreted by endothelium triggers “bystander” gemcitabine uptake via ENT1 in tumor cells. As such, these investigations potentially define failure to stimulate ASMase/ceramide signaling as mediating a new form of chemoresistance. It is anticipated that information derived from studies proposed here will inform a planned follow up clinical trial for advanced sarcoma to be performed by the Sarcoma Service at MSK.

NIH Research Projects · FY 2026 · 2020-12

PROJECT SUMMARY/ABSTRACT While checkpoint inhibitors and chimeric antigen receptor (CAR) T cells undergo widespread investigation as approaches to unleash the immune system’s tumor-targeting abilities, the mechanisms by which these therapies fail is the subject of great debate. In the setting of solid tumors, it is believed that the microenvironment is hostile, excluding T cells and/or inhibiting their ability to proliferate or be activated. Metabolic precursors, most notably glucose, has been implicated as inhibiting T-cell function. There remains an unmet need for approaches to better understand T-cell metabolism and its impact on tumors in vivo, as well as a method to modulate this metabolic limitation to overcome T-cell exhaustion. Given extensive progress through our original award and additional preliminary data, we have developed a platform to allow T cells to overcome the nutrient limitation of the tumor microenvironment by utilizing the fructose from our diets to power metabolism. Moreover, we have developed novel methods to trace metabolism in vitro and in vivo using hyperpolarized magnetic resonance (HP MR), which can detect changes in metabolism in real time. Taken together, these approaches provide a platform for studying immunometabolism both in vitro and in vivo, which has great potential for future immunotherapeutics. The objective of this innovative renewal proposal is to utilize our platform to develop physiologically relevant fructose enabled CAR-T cells and visualize their metabolism in vivo. In Aim 1, we will develop CAR-T cells that target a highly expressed cancer antigen Epha2 and use fructose to boost their metabolism. In Aim 2, we will expand our repertoire of metabolic imaging strategies to trace all of fructolysis with hyperpolarized magnetic resonance. Given that our fructose metabolic engineering can drive glycolytic flux, in Aim 3 we will explore further pushing this metabolism by combining it with novel approaches to funnel lipids into the TCA cycle, thus creating a paradigm shifting approach to cancer immunotherapy. It is the overarching goal of this renewal proposal to build on the novel approaches in metabolism and metabolic imaging we pioneered and to further our understanding of immunometabolism to transform immunotherapy strategies in patients.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT The incidence of esophagogastric (EG) cancer is increasing rapidly, notably among young men. In patients clinically classified as HER2-positive (ERBB2 amplification and/or 2+/3+ protein overexpression), the combination of the therapeutic anti-HER2 antibody trastuzumab and standard cytotoxic therapy prolongs progression-free and overall survival. However, intrinsic tumor resistance or mechanisms of resistance developed during treatment limit the clinical benefit in 32% of patients, and other anti-HER2 therapeutic antibodies failed in clinical trials to treat EG cancer. Complementary biomarkers and methods are therefore needed to treat such patients. Guided by preclinical data suggesting that caveolin-1 (CAV1) – the main protein of cholesterol-rich invaginations of the plasma membrane – reduces trastuzumab binding to HER2-positive EG tumors, we initiated retrospective clinical analyses to validate CAV1 as a complementary biomarker of HER2. Remarkably, Kaplan-Meier survival analyses demonstrated that HER2+ EG tumors expressing high CAV1 (IHC 2+/3+) had worse overall survival than those expressing low CAV1 (IHC 0/+1) after trastuzumab therapy. These promising preliminary results prompted us to pharmacologically deplete CAV1 (which is present in cholesterol membrane domains) with lovastatin, a cholesterol-depleting drug. Here, we will perform retrospective analyses of patients with HER2-positive EG tumors to assess HER2 expression and heterogeneity, ERBB2 amplification, CAV1 staining and the presence of genetic alterations (copy number variations) associated with trastuzumab resistance. We will analyze medical records to determine if concurrent statin use is associated with enhanced response to trastuzumab. In addition to retrospective analyses, we will perform randomized imaging and therapeutic preclinical studies using patient-derived EG xenografts (PDXs) representing HER2+/CAV1High and HER2+/CAV1Low tumor populations. We will determine the molecular imaging profile (89Zr-Trastuzumab PET) and therapeutic efficacy in PDXs treated with (1) control saline, (2) trastuzumab alone, (3) lovastatin alone, or (4) the combination of trastuzumab with lovastatin, to identify molecular features that confer drug sensitivity and resistance to this promising investigational combination. Aim 1 will validate CAV1 as a complementary biomarker to HER2, Aim 2 will determine the potential dosimetric impact of the statins on clinical imaging and identify EG tumor populations that benefit from the trastuzumab/lovastatin combination, and Aim 3 will validate the use of a statin as a new pharmacologic approach to HER2-targeted imaging and systemic radionuclide therapy (endoradiotherapy) capable of reducing off-target radiation doses. All three aims will generate important new preclinical data on the use of statins to improve trastuzumab efficacy, which should provide an excellent foundation for many future investigations, including clinical translation of trastuzumab/statin combination therapy and potential broader application to other HER2+ cancers. The long-term translational objectives are to establish the foundation for a clinical trial combining statin with trastuzumab to prevent or delay the emergence of drug resistance in patients with HER2+ EG cancer.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Gynecologic cancers are some of the most lethal diseases affecting women. Globally, one woman dies of cervical cancer every two minutes. MRI is increasingly used in the evaluation of gynecologic and many other cancers. Beyond its established use for cancer staging, there has long been an interest in the use of MRI-derived quantitative metrics to gain insights into the tumor microenvironment. Parametric maps obtained from quantification of dynamic contrast enhanced (DCE) MRI data can be used to study tumor vascularity and identify tumors that are better perfused and oxygenated and thus more sensitive to some treatments such as chemotherapy and radiation. However, the relative slow imaging speed and motion sensitivity of current MRI technology results in non-reliable and non-reproducible quantification of DCE-MRI data, which restricts its application in clinical practice. Our group is a world leader in development of rapid motion-resistant DCE-MRI techniques, in particular using combinations of radial imaging and compressed sensing. We developed the technique called GRASP, which was conceived as an academic-industrial partnership and has now been successfully translated into standard clinical practice. Though powerful, the first generation of GRASP has limitations. First, radial imaging is robust to motion, but not free of motion, which usually results in blurring. Second, GRASP uses a very simple sparsifying transform for compressed sensing, which can introduce issues with quantification. Third, GRASP was not originally developed for pharmacokinetic analysis and misses important ingredients such as integration of AIF estimation and T1 mapping. Fourth, image reconstruction time is still very long – in the order of several minutes. We have developed new advances to circumvent these limitations and offer a new DCE-MRI technique with increased speed, motion-resistance and personalized AIF estimation and T1 mapping for pharmacokinetic analysis. Following the PAR-18-009 guidelines, our main goal is to form an academic-industrial partnership between Memorial Sloan Kettering Cancer Center and General Electric Healthcare to translate these new developments in quantitative DCE-MRI for use in patients with gynecologic and other type of cancers. Specific Aims are as follows: 1. Develop and implement a fast motion-resistant quantitative DCE-MRI technique that goes beyond GRASP to offer increased speed and resistance to motion; dynamic T1 mapping; and personalized and automated pharmacokinetic analysis 2. Evaluate the repeatability, reproducibility and preliminary tumor response assessment of the fast motion- robust quantitative DCE-MRI technique (“DCE-new”) and compare DCE-new to standard of care DCE-MRI (“DCE-standard”) in patients with gynecologic cancer 3. Develop and evaluate fast image reconstruction algorithms based on deep learning

NIH Research Projects · FY 2025 · 2020-09

Efficient yet effective models for delivering genetic counseling and testing are sorely needed to meet increasing demands for timely genetic risk information. Traditional germline genetic testing models, which include in-depth genetic counseling both before and after testing, are time intensive and place substantial demands on the limited genetic counselor workforce. A “mainstreaming” model, which allows for non-genetics healthcare providers to order genetic testing without pre-test genetic counseling, with support from genetic counselors at the time of result disclosure, has shown promise. Yet, past evaluations of mainstreaming models have been hampered by serious limitations: Studies were restricted to the context of BRCA1/2 testing and do not reflect the growing use of multigene panel testing (MGPT); rarely used rigorous experimental study designs or evaluated theoretically-relevant decision-making, psychosocial, and communication outcomes; have not capitalized on opportunities to improve post-test clinical and familial communication; and neither included nor addressed informational needs of different kinds of patients. We overcome these limitations with the proposed study, the objective of which is to develop, test, and evaluate a mainstreaming model for hereditary cancer MGPT among cancer patients of many different groups. We will first use formative research methods, including transcreation and cognitive interviewing, to adapt existing pre-test educational materials and post-test clinical communication materials for use among the many different types of patients treated at our local hospital partnering sites. Next, we will conduct a randomized controlled trial (RCT) involving patients diagnosed with breast, colorectal, ovarian, pancreatic, or prostate cancer (N=350). Patients will be randomized to obtain access to cancer MGPT through either: i) standard-of-care wherein in-depth pre-test and post-test genetic counseling are provided via telegenetics (i.e., videoconferencing delivered at the site clinic) with standard post-test clinical communication materials, or ii) mainstreaming intervention wherein patients receive the adapted pre-test educational materials with testing ordered by their oncologist, followed by post-test genetic counseling provided via telegenetics with adapted clinical communication materials. Patients will complete assessments of decision-making, psychosocial, and behavioral outcomes at baseline, upon deciding whether to have MGPT, and at 1-week and 6-months following receipt of their test results. This research has the potential to transform genetic counseling and testing practice by ensuring effective risk communication, decision making, and uptake of genomic medicine to by all the groups of patients with hereditary cancer syndromes.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Transcriptional dysregulation in tumors can induce the abundant expression of repetitive elements in cancerous cells compared to normal tissues, where they are often transcriptionally silent. Such transcripts have been associated with better outcomes to cancer immunotherapies, as they can modulate the tumor immune microenvironment and generate an under-quantified source of tumor neoantigens. Therefore, it has been hypothesized that the aberrant transcription of repeat RNA is both a critical mechanism for initiating the immune response in the tumor microenvironment and an untapped source of potential therapeutic targets. Using a set of approaches from statistical physics, our team predicted repetitive element RNA directly stimulates receptors of the innate immune system, confirmed this hypothesis in a key subset of immune cells, and showed repeat expression can correlate with response to checkpoint blockade immunotherapies. Repeat RNA is therefore both a novel biomarker for the innate immune response in cancer and a potential therapeutic target to modulate tumor immunity. We will utilize a set of tools, developed by our team, from statistical physics to characterize repeat RNA recognition by innate immune receptors in silico and their role in tumor-immune co-evolution, both with and without the application of immunotherapy (Aim 1). Next, we will characterize the spatial context of repeat RNAs in the tumor immune microenvironment and the co-localization of predicted immunostimulatory RNA with activation of immune signaling, along with in depth immune-phenotyping of the state of the immune microenvironment in vivo (Aim 2). Finally, we will perform functional validation of our predictions on human immune cells to validate mechanisms of recognition and the specific immune subsets responsible for repeat recognition via a set of in vitro assays (Aim 3). Our goal is to use approaches from statistical physics to quantify the role of repetitive elements in tumor immunology, their rules of recognition by innate immune receptors and their part in facilitating cytolytic T cell activity. In doing so we will combine novel RNA detection technologies to study their spatial distribution and localization in cancers; state of the art immune-phenotyping; and mathematical models to characterize their direct role in tumor evolution. We hypothesize that our approach from statistical physics will identify the key structural and sequence features of repeat mediated immune activation in solid tumors and shed light on their specific consequences for tumor evolution and therapeutic efficacy.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Enhancers are essential regulatory elements that together with transcription factors (TFs) instruct cell- type specific transcriptional programs during development, tissue homeostasis and regeneration. Initiatives such as the ENCODE project, revealed tens of thousands putative enhancers based on linear proximity, using criteria like chromatin accessibility, TF binding, and histone modifications such as H3K27ac. However, a main challenge of uncovering functional enhancers and assigning them to target genes lies in the complexity of the 3D chromatin organization, which can influence enhancer specificity and activity. Using an advanced chromosome conformation capture assay, we recently captured the dynamic rewiring of 3D enhancer networks during mouse somatic cell reprogramming and discovered multi-connected enhancers that we named “3D enhancer hubs”. Here we extend the 3D mapping approach to human primary islets, and compare islets from healthy and type 2 diabetes (T2D) donors to assemble a 4D atlas to capture the rewiring of 3D enhancer network in disease progression. At the same time, we plan to compare the enhancer network in adult islets to earlier stages of development by using human pluripotent stem cells (hPSCs) to generate early β cells and their developmental precursors. Utilizing these 4D genomic data, we will computationally nominate core β-cell specific enhancers relevant to β cell development, function, and T2D, and then interrogate these putative enhancers through large-scale CRISPRi mediated perturbation screens using hPSC-β cells. Enhancers identified from the screening effort will be further validated in an established human β cell line and primary human islet β cells. This proposal addresses a critical gap in the 4DN initiative, that is how to translate 3D genomics data into functional data with respect to gene expression in the context of human health. Successful completion of our aims will establish a paradigm for the discovery and interrogation of functional enhancers that instruct transcriptional programs specific to a cell type of interest, reveal unique insights into their mechanisms of action, and identify enhancers with relevance to human development and disease. For instance, uncovering functional enhancers could assist the identification of noncoding causal variants identified in genome-wide association studies.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Intrahepatic cholangiocarcinoma (IHC) is a subtype of biliary tract adenocarcinoma with a poor prognosis and rising incidence. SEER data recently documented an average 4.4% rise over the past decade. Similarly, an analysis spanning 18 years reported a 7% annual increase in incidence, the highest of any biliary tract subtype. Both studies highlight the abysmal survival of patients with IHC, the latter reporting a median of approximately 6 months. Most patients with IHC have unresectable disease at diagnosis, and even those few fortunate enough to undergo resection recur commonly. For these patients, treatment options are limited, with systemic chemotherapy representing the standard, and in most cases, the only approach. Combination therapy with gemcitabine (GEM) and a platinum agent is the current gold standard, but its benefit is limited, offering a median overall survival of approximately 12 months. Recently, our group completed a phase II single-arm study of regional chemotherapy using continuous infusion hepatic arterial (HAI) floxuridine (FUDR) combined with GEM and oxaliplatin (OX). The median overall survival was 25 months, with four patients responding sufficiently to undergo resection. Based on these promising results, the primary goal of this proposal is to establish the efficacy of HAI FUDR added to the most active systemic regimen (GEM/OX) for the treatment of unresectable IHC in a multi-center randomized phase II study, with the primary endpoint of progression-free survival. Further improvements in the management of IHC have been hindered in large part by a poor understanding of the biology of the disease. IHC is among the most genomically heterogeneous solid tumor, resulting from the wide array of risk factors and multiple potential cells of origin. Significant heterogeneity has been shown not only from patient to patient, but even within the same tumor. This feature has precluded precise characterization of molecular pathogenesis, made it difficult to identify effective targeted therapies, and results in inaccurate assessment of the mutational landscape when based on a single biopsy. The proposed clinical trial provides an ideal opportunity to address these gaps. We will evaluate intratumoral heterogeneity (ITH) using targeted exome sequencing of multiple tumor biopsies and cell-free DNA (cfDNA) from a large cohort of patients and use these findings to stratify patients with respect to response and survival. For the subset of patients that progress while enrolled in the trial, we will obtain tumor biopsies and blood at the time of progression to elucidate mechanism(s) of treatment resistance by tumor DNA and cfDNA sequencing. Using these same samples, we will establish patient-derived organoid models for functional evaluation of genetic predictors of treatment response. Finally, we will use radiogenomics, or the merger of quantitative imaging with molecular analyses, to explore non- invasive stratification of patients based on mutational patterns and to quantify the degree of heterogeneity. Using an integrated analysis approach, these studies will provide a foundation for multimodal risk stratification for IHC.

NIH Research Projects · FY 2024 · 2020-09

Summary The analysis of liquid biopsy (eg, cell-free DNA [cfDNA]; circulating tumor cells [CTC]) in blood is increasingly integrated in clinical contexts including diagnosis, disease monitoring, understanding resistance, and early detection of relapse. The key challenge of detecting these analytes is that they are present at a very low proportion of the biospecimens, and therefore are heavily influenced by pre-analytical factors associated with acquisition and processing. Understanding effects of these pre-analytical variables on the quality of data generated in downstream molecular CTC and cfDNA assays is critical for robust clinical implementation of liquid biopsy tests. To date, research efforts have focused on effects of preservation methods, processing time, storage temp, and shipment conditions on quality of CTC and cfDNA in blood plasma. There are no studies reported on effects of patient-specific context such as fasting, administration of anti-emetics, or biospecimen acquisition procedures (eg, order of blood collection aliquots, time of day when blood is drawn, etc.) There is a lack of data on this type of pre-analytical variable that impacts design of clinical trials such as optimal timing for blood draw and interpretation of data to distinguish technical variables introduced by these pre-analytical factors from the biological signals being evaluated. We propose to address this gap by extending the work done by our team members on evaluating effects of sample processing protocols on cfDNA and CTC analysis, to further investigate effect of patient-specific context. Hypothesis: Pre-analytic variables may affect signal-to- noise ratio in cfDNA and CTC analysis and thus have a higher impact on quantification at levels close to the assay limit of detection. Aim 1: Determine the effect of patient-specific context on the quality of cell-free DNA (cfDNA) and circulating tumor cells (CTC) in prostate cancer patients. Aim 2: Evaluate the impact of these variables on the performance of downstream cfDNA and CTC molecular profiling assays. We will apply an adaptive design in which we perform initial analysis with 20 patients per cohort, then adjust as needed. In a foundation-funded pilot study, we focused on one cohort to study effect of draw order. Results confirm the variability of biomarkers quantification as a result of pre-analytical variables. Significance: Results will elucidate effects of multiple pre-analytical variables specific to individual patient context on performance of blood-based biomarker analysis in cfDNA and CTC. These data will inform the design of liquid biopsy-incorporated clinical trials by identifying optimal timing of blood collection to minimize effects of pre-analytical variables. Innovation: This will be the first study to examine the effect of patient- specific context on quality of liquid biopsy data. We will collaborate closely with commercial liquid biopsy test developers as part of the Blood Profiling Atlas in Cancer (BloodPAC) project, with the goal of sharing knowledge across different sectors and working toward harmonization of pre-analytical procedures for liquid biopsy testing.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Phenotypic plasticity and its regulation by contextual signals is of central importance to tumor biology. TGFβ is a major regulator of cell phenotype during development, tissue homeostasis, regeneration, and cancer. Our long- term goal is to elucidate TGFβ signaling and the principles that govern its effects on normal and neoplastic cells. This proposal is based on our long-standing contributions to delineating the TGFβ signal transduction pathway, its context-dependent effects, and its aberrant activity in tumorigenesis and metastasis. The proposed work builds on recent progress towards understanding how TGFβ-activated SMAD transcription factors regulate differentiation in stem and progenitor cells (Aragón et al Genes Dev. 2019; Wang et al Cell Stem Cell 2017), the basis for TGFβ-mediated tumor suppression and the evasion of this effect (David et al Cell 2016; Huang et al Cancer Disc. 2019), and the development of experimental models of dormant metastasis to expose the role of TGFβ in this poorly understood, yet highly significant aspect of cancer (Malladi et al Cell 2016). Moreover, we recently elucidated how TGFβ triggers epithelial-mesenchymal transitions (EMTs) in pancreatic ductal adenocarcinoma (PDA), lung adenocarcinoma (LUAD), and embryonic stem (ES) cells, and how these phenotypic plasticity events are coupled either to fibrogenesis or to differentiation depending on the epigenetic context (Su et al Nature 2019). Based on these advances and unique experimental models and human tumor single-cell analytics that we have developed, we will address long-standing questions of growing importance: How does TGFβ signaling regulate epithelial cell plasticity in development and cancer? What is the role of TGFβ-induced intra-tumoral fibrosis during tumorigenesis? What is the relevance of this mechanism to TGFβ-induced organ fibrosis? How does TGFβ drive metastasis-initiating cells into EMT-linked growth arrest? Does this state render cancer cells immune-evasive during metastasis dormancy? To investigate these questions, we will dissect an obscure RAS effector, RREB1, which we recently identified as a key partner of TGFβ-activated SMAD transcription factors in the induction of fibrogenic and developmental EMTs. We will elucidate the role of EMT-linked intra-tumoral fibrosis in tumor growth and metastasis. Focusing on metastasis- initiating cells, we will follow recent evidence that TGFβ imposes a quiescent, immune evasive state that provides long-term survival to dormant metastasis cells and potentially resistance immunotherapy. Collectively, these studies will provide knowledge and experimental models to delineate the role of TGFβ in fibrosis, tumor invasion and metastasis, and will better define how and when to target TGFβ in cancer.

NIH Research Projects · FY 2025 · 2020-09

Towards Targeting the Follicular Lymphoma Microenvironment Follicular lymphoma (FL) is the second most common and still incurable form of B cell lymphoma. FL is a slow growing cancer that shows a unique dependence on a supportive microenvironment. The supportive niche also affects FL therapies. For example, inhibitors of BTK, PI3K, or BCL2 show exciting activity against aggressive lymphomas and chronic leukemia, but they have little activity against indolent FLs. We speculate that the FL microenvironment protects and sustains the malignant B cells and contributes to FL development, progression, and resistance to therapy. Conversely, we propose that disrupting interactions in the FL niche will be especially effective against FL. Our hypothesis is based on prior work by others and our own work on immune receptor mutations in FL (e.g. TNFSRF14 and EphA7) have revealed cell-cell interactions as key drivers and sustainers of FL biology. My lab has made major contributions to our understanding of the biology and genetics of FL. We built an accurate mouse model of FL and we and others have used this model to interpret the biological function of the most common FL drivers. For example, we reported on the role of epigenetic driver mutations in KMT2D, CREBBP, EZH2, we investigated FL cell metabolism, cell cycle control, aberrant mRNA translation, and the outstanding importance of immune receptors (e.g. TNFSRF14, b2M, EphA7) in FL biology. This work has been reported in an extensive series of high-impact publications. It has also led to several patent filings that protect experimental lymphoma therapies. We now propose a systematic assessment of the cellular composition of the FL microenvironment using cutting edge single-cell RNA sequencing on murine and human FLs. We want to understand how tumor genotype, indolent versus transformed disease stage, and therapy (esp. checkpoint inhibition) shapes the FL niche. We use single cell data to formulate specific hypotheses concerning cell-cell interactions and we use genetic and molecular biology tools to explore underlying mechanisms. Our goal is to identify opportunities to disrupt the supportive niche and to exploit this for new therapies. We have already had some success in this regard, and we engineered bi-functional antibodies and modified CAR-T cells that target interactions between malignant B cells and supportive niche elements. !

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Somatic mutations in the isocitrate dehydrogenase (IDH) enzymes contribute to the pathogenesis of acute myeloid leukemia (AML) and other malignancies via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). D-2HG blocks differentiation of malignant cells by inhibiting alpha-ketoglutarate (KG)-dependent enzymes that regulate chromatin structure and gene expression. Small molecule inhibitors of mutant IDH enzymes are promising new therapies for AML, but their efficacy remains limited to the subset of patients with IDH mutations. This raises the question as to whether analogous metabolic aberrations might contribute to leukemogenesis in IDH-wildtype AML. Intriguingly, 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH mutants exclusively produce D-2HG, biochemical studies indicate that L-2HG can function as a ~10-fold more potent inhibitor of many KG-dependent enzymes, including chromatin modifiers and regulators of hypoxia-inducible factor (HIF) stability. However, biological sources and activities of L-2HG have been poorly understood. We identified a metabolic pathway wherein normal and malignant cells without IDH mutations selectively produce L-2HG in response to oxygen limitation (a.k.a. hypoxia) through an unusual reaction catalyzed by lactate dehydrogenase (LDHA). We show that hypoxia-induced L-2HG enhances stability of HIF, increases repressive chromatin modifications, and blocks differentiation of stem/progenitor cells. These findings suggest that L-2HG might account, at least in part, for the importance of hypoxic niches, HIF, and LDHA in balancing self-renewal and differentiation of stem cell populations, including hematopoietic stem/progenitor cells (HSPC) and leukemia stem cells. Thus, we hypothesize that L-2HG functions as a metabolic signal that couples hypoxic niches to the maintenance of normal blood stem cells and leukemia stem cells. This hypothesis will be rigorously addressed in three Specific Aims. Aim 1 will define the molecular mechanisms by which L-2HG regulates blood cell differentiation in vitro. In this Aim, we will define the effects of L-2HG on gene expression and chromatin structure and determine how these inputs balance HSPC stemness and lineage differentiation. Aim 2 will determine how L-2HG functions to control normal and malignant hematopoiesis in vivo. This Aim will use novel genetically engineered mouse models that allow for tissue-specific, inducible manipulation of L-2HG levels in order to dissect the role of L-2HG in normal hematopoiesis and leukemia. Aim 3 will elucidate the oncogenic mechanisms and therapeutic potential of L-2HG in human leukemia. In this Aim, we will use primary AML biospecimens and patient-derived xenografts to determine the mechanisms that lead to deregulated L- 2HG in a subset of AML and assess whether depleting L-2HG offers a promising strategy to treat human AML. The proposed studies will offer fundamental insights into the metabolic control of normal and malignant stem cell biology and expand the applicability of metabolic targeted therapies for leukemia and other cancers.

NIH Research Projects · FY 2025 · 2020-08

Abstract The goals of my research since 1978 have been to distinguish the features of cancer cells from healthy cells in order to be able to discover and develop safe and selective, innovative immunotherapies. Here, we leverage my past body of work that has evolved from native mouse antibodies, to humanized mAb, to various potent conjugates of these mAb, to TCRm antibodies, and ultimately to BiTE forms and CAR forms to create the latest generation of agents and experiments now proposed. This scientific progression has been sustained for more than 3 decades. This work is innovative, as noted by our numerous therapeutic firsts and more than 3 dozen patents, including: human antibodies for the treatment of acute leukemia, targeted alpha- particle therapies, in vivo alpha-particle isotope generators, oncogenic fusion point vaccines, human TCR mimic antibodies to intracellular oncogenic proteins, and most recently, various innovative CAR technologies, now in progress. Several of the antibodies and vaccines reached late stage, national clinical trials such as a WT1 vaccine, Galenpepimut, and our alpha generator-Lintuzumab. But now, how do we achieve true cancer specificity? The immune system has evolved the T cell and TCR as a highly efficient and truly selective system capable of recognizing viral and mutated intracellular proteins derived from inside the cell. Therefore, in this OIA the questions are: Is it possible to make truly cancer selective monoclonal antibodies, and various derived molecular platforms, that will be effective therapeutically by mimicking a TCR? What are the obstacles and cancer resistance mechanisms to this approach and how will they be overcome? How do we select the right target epitopes and also avoid inevitable off- targets that may cause toxicity? The following issues will be addressed: A. Target choices: What are the best epitopes from a biochemical, biophysical, or immunological point of view? Are certain classes of proteins or structures of peptides preferred? How do we design screens for TCRm? B. Can we modulate the expression of the epitopes or the antigen presentation machinery? How is the MHC ligandome generally affected by these drugs and is this important? C. Predictive tools: Can we develop proteomic and genetic tools to create general rules and to help guide us to picking epitopes and predicting which may be safe? D. What cancer therapeutic platform for the TCRm makes the most sense in light of what we have learned about the biology and immunology of the epitope, as well as the predictions of specificity from the tool sets?

NIH Research Projects · FY 2025 · 2020-08

ABSTRACT The research projects proposed in this SPORE address genomic instability in breast cancer. Three areas are the focus of study: homologous recombination deficiency, chromosomal instability and APOBEC mutagenesis. Our ultimate plan is to exploit tumor specific vulnerabilities by virtue of their underlying genomic instability. These profiles of genomic instability have offered novel insights about the drivers breast cancer development and progression. There are opportunities for therapeutic advances in breast cancer, which have emerged based on the initial successes, for example, in utilizing homologous recombination deficiency by treatment with a PARP inhibitor. The plan is to determine the optimal use of these agents and develop novel agents for these tumors. Chromosomal instability, which does not necessarily have a unique pattern of mutations, is associated with a poor prognosis, but no specific therapeutic strategy at present. The link between chromosomal instability and innate immune signaling has been made, and the goal is to exploit this connection for therapy. For APOBEC, we know that a characteristic pattern of SNVs are observed, but in this application, we are highlighting the role of APOBEC in the acquisition of drug resistance, and introducing novel approaches for reliably identifying and therapeutically targeting breast cancers with an active APOBEC mutagenesis process. In summary, the goals are to take the risks of genomic instability (poor prognosis, rapid development of resistance) and turn genomic instability into an advantage for therapeutic targeting, thereby improving the prognosis for high risk breast cancers.

NIH Research Projects · FY 2025 · 2020-08

Project Summary/Abstract Homologous recombination, i.e., homology-directed repair (HDR), is a major repair pathway for double-strand breaks (DSBs), including lesions arising during DNA replication. HDR mutants are characterized by genomic instability and sensitivity to DNA damaging agents such as interstrand cross-linking agents like cisplatin and poly(ADP-ribose) polymerase inhibitors, both of which are used in cancer treatment. Several proteins central to the HDR pathway are tumor suppressors, notably the breast and ovarian cancer suppressor BRCA2, which promotes the function of RAD51, the critical protein for homologous strand exchange. RAD51 paralogs are also key HDR proteins and have also been identified both as tumor suppressors and as proteins that affect therapy response. These HDR proteins are essential. Individuals with germline mutations are constitutionally heterozygous, but tumors typically have somatic undergone loss of heterozygosity (LOH), losing the wild-type allele, presumably as an early step in tumor initiation. This proposal has an overarching goal of integrating our understanding how HDR proteins act to maintain genomic stability and cell and tissue homeostasis, how they come to be “lost” in cells, and how their function can be restored. Thus, this broad goal impacts tumor initiation, therapy response, and therapy resistance. It incorporates molecular analysis of HDR protein function, with a particular focus on BRCA2, and delineates how cells respond to HDR protein loss, including how they escape cell death to allow tumor formation. Within this goal is understanding tumor initiation from the standpoint of determining mechanisms of LOH that lead to HDR protein loss, as well as uncovering factors that affect LOH frequencies. While HDR protein loss sensitizes tumors to targeted therapies, HDR function is often restored by secondary mutations, leading to therapy resistance. Understanding which mutations are susceptible to reversion and how therapy impacts reversion is of major interest. Finally, HDR within tissues is also part of this integrated goal, in particular, within the fallopian tube epithelium, which is considered the tissue of origin of high-grade serous ovarian cancers.