MYC-driven Tumors Research Articles

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

MDB-52. MYC-DRIVEN PEDIATRIC BRAIN TUMORS SHARE A COMMON PHOTORECEPTOR IDENTITY

Abstract Mouse models serve as invaluable tools for improving the understanding and treatment of pediatric brain tumors. We recently published a highly aggressive MYC-driven brain tumor model (GMYC), which exhibits approximately 70% penetrance. Our transcription profiling indicates that GMYC tumors accurately resemble human Group 3 medulloblastoma (MB-G3). Interestingly, a strong photoreceptor program was activated in the tumor model. This is commonly upregulated in MB-G3 but also in pineoblastoma (PB), another malignant pediatric brain tumor. CRX, a transcription factor critical for pineal cell development is also a master regulator for MB-G3 maintenance. By studying early tumor initiation using Crx-lineage tracing and scRNA-seq, we confirmed that GMYC tumors originate from early pinealocytes. Exome sequencing of GMYC tumors further confirmed mutations of epigenetic regulators commonly reported in either PB or MB. To investigate tumor development from photoreceptor-positive progenitors in the developing brain, we performed Crx-lineage tracing with tamoxifen injections, confirming that Crx-positive cells exist in pineal gland progenitors. However, Crx-traced cells also marked granule neurons in the flocculonodular lobe of the cerebellum, which is the suggested site of MB-G3 origin arising from the developing rhombic lip. To investigate if the Myc oncogene can generate different types of pediatric brain tumors in Crx-positive cells, an XMYCT58A-Tomato mouse model was established by crossing Crx-CreERT2; LSL-MycT58A and Rosa26LSL-tdTomato strains. Here, MycT58A was turned on with tamoxifen injection after birth and tumor development followed using the tdTomato reporter. XMYCT58A-Tomato mice developed brain tumors from both the pineal gland and cerebellum after 2-6 months. Histologically, tumors were non-glial (GFAP-, Olig2-), showed neuronal activity (Neurod1+), and stained positive for photoreceptor identity markers (CRX+, OTX2+) similar to both MB-G3 and PB-MYC. Collectively, our data suggest that both MYC-driven MB-G3 and PB-MYC originate from photoreceptor-positive cells, which has implications for developing novel treatments targeting these devastating childhood malignancies.

MDB-88. DUAL TARGETING OF PROCESSIVE TRANSCRIPTION FOR MYC-DRIVEN CIRCUITRY IN MEDULLOBLASTOMA

Abstract BACKGROUND The highest-risk medulloblastomas are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB. METHODS Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with the dual CDK9/7 inhibitor zotiraciclib to evaluate effect on Myc-driven programs and tumor growth. RESULTS Eight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cell lines are sensitive to CDK9 inhibition at low nanomolar concentrations, and these synergize with CDK7 inhibition. We find that treatment with the dual CDK9/7 inhibitor zotiraciclib depletes Myc binding and transcriptional activity at enhancer elements within the Myc topologically associated domain, largely abrogating transcriptional output from the Myc promoter. This leads to a decrease in Myc binding genome-wide with a concordant downregulation of hallmark Myc-driven transcriptional programs. Clinically relevant CDK9 inhibitors show variable efficacy in vivo, but CNS-penetrant agents achieved a significant prolongation in xenograft survival. CONCLUSIONS CDK9/7 catalytic activity represents a druggable vulnerability underpinning Myc-driven transcriptional programs. The development of CNS-penetrant CDK9/7 inhibitors may open new avenues for rational therapy in these high-risk medulloblastomas.

Tryptophan fuels MYC-dependent liver tumorigenesis through indole 3-pyruvate synthesis

Cancer cells exhibit distinct metabolic activities and nutritional dependencies compared to normal cells. Thus, characterization of nutrient demands by individual tumor types may identify specific vulnerabilities that can be manipulated to target the destruction of cancer cells. We find that MYC-driven liver tumors rely on augmented tryptophan (Trp) uptake, yet Trp utilization to generate metabolites in the kynurenine (Kyn) pathway is reduced. Depriving MYC-driven tumors of Trp through a No-Trp diet not only prevents tumor growth but also restores the transcriptional profile of normal liver cells. Despite Trp starvation, protein synthesis remains unhindered in liver cancer cells. We define a crucial role for the Trp-derived metabolite indole 3-pyruvate (I3P) in liver tumor growth. I3P supplementation effectively restores the growth of liver cancer cells starved of Trp. These findings suggest that I3P is a potential therapeutic target in MYC-driven cancers. Developing methods to target this metabolite represents a potential avenue for liver cancer treatment.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

PRIM2: A Marker of MYC-driven Hyper-proliferation, Disease Progression, Tumor Aggressiveness and Poor Survival in Glioma Patients.

Gliomas are the most prevalent brain tumors with metabolic alterations playing a pivotal role in disease progression. However, the precise coordination of metabolic alterations with tumor-promoting cellular mechanisms, leading to tumor initiation, progression, and aggressiveness, resulting in poor outcomes, remains poorly understood in gliomas. We conducted a metabolism-targeted differential gene expression analysis using glioma patients' expression profiling data from The Cancer Genome Atlas (TCGA) database. In addition, pathway enrichment analysis, gene set enrichment analysis (GSEA), transcription factor prediction, network construction, and correlation analyses were performed. Survival analyses were performed in R. All results were validated using independent GEO expression datasets. Metabolism-targeted analysis identified 5 hits involved in diverse metabolic processes linking them to disease aggressiveness in gliomas. Subsequently, we established that cell cycle progression and hyper-proliferation are key drivers of tumor progression and aggressiveness in gliomas. One of the identified metabolic hits, DNA primase 2 (PRIM2), a gene involved in DNA replication was found directly associated with cell cycle progression in gliomas. Furthermore, our analysis indicated that PRIM2, along with other cell cycle-related genes, is under the control of and regulated by the oncogenic MYC transcription factor in gliomas. In addition, PRIM2 expression alone is enough to predict MYC-driven cell cycle progression and is associated with tumor progression, aggressive disease state, and poor survival in glioma patients. Our findings highlight PRIM2 as a marker of MYC-driven cell cycle progression and hyper-proliferation, disease onset and progression, tumor aggressiveness, and poor survival in glioma patients.

Abstract A061: A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations

Abstract One of the most aggressive cancers in the US is pancreatic ductal adenocarcinoma (PDAc), whose incidence is expected to rise in the coming years. The standard-of-care treatments have improved over the past few decades, but they still primarily involve harsh chemotherapy cocktails and have only marginally increased overall survival, to a 5-year survival rate of 12 percent. c-MYC (MYC), a potent transcription factor, is frequently dysregulated in PDAc. In PDAc and other cancers, MYC plays a significant role in tumor progression, and its dysregulation has been linked to tumor aggressiveness and therapeutic resistance. In various mouse models of cancer, it has been demonstrated that oncogenic MYC expression controls components of the tumor microenvironment (TME). MYC expression in PDAc has been associated with a desmoplastic immune suppressive TME, though the precise mechanism is still unknown. In this study, we introduce a novel genetically modified mouse model (GEMM) of PDAc that can be used to simulate the disease's heterogeneity and investigate how MYC controls the tumor’s immune and stromal microenvironments. In the pancreas of our KMCERT2 model, both mutant Kras and lowly deregulated Myc are expressed through inducible Cre-driven mechanisms. Our research demonstrates how deregulated MYC cooperates with oncogenic KRASG12D in the adult pancreas to promote PDAc, faithfully reproducing the inter- and intra-tumoral heterogeneity observed in clinical PDAc populations. Currently, an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC) is used for the majority of murine studies of PDAc. To learn more about the mechanisms underlying observed phenotypes in our KMCERT2 model, RNA- and DNA sequencing were performed on microdissected autochthonous tumor specimens as well as KMCERT2 tumor-derived cell lines. In contrast to the KPC model, our inducible KMCERT2 model of PDAc exhibits genetic alterations, such as CDKN2A loss, comparable to human disease. A distinct loss of nuclear p53 expression is also observed. Intriguingly, multiplexed immunohistochemistry analysis of immune cell composition showed that MYC-driven tumors have a higher density of antigen-presenting cells (APCs) when compared to spontaneous KPC tumors. Standard-of-care medications like gemcitabine and FOLFIRINOX are frequently ineffective against human PDAc. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines show a variety of responses to these therapies, with both resistant and sensitive tumor models, allowing us to use this model to better understand the mechanisms causing therapeutic resistance and to test new therapies. Additionally, we observe consistent liver metastasis in both spontaneous and orthotopic transplant settings. Together, these studies investigate the function of dysregulated MYC expression in murine PDAc metastatic behavior, immune phenotypes, and therapeutic response. It also offers mouse models of PDAc that are both spontaneous and orthotopic, recapitulating the complex and highly metastatic nature of the human disease. Citation Format: Isabel A. English, Patrick J. Worth, Kevin A. MacPherson, Michael B. Heskett, Vidhi Shah, Jackie L. Phipps, Courtney Betts, Shamilene Sivagnanam, Carl Pelz, Amy S. Farrell, Brittany L. Allen-Petersen, Meghan M. Joly, Motoyuki Tsuda, Colin J. Daniel, Jenny Eng, Xiaoyan Wang, Ashley L. Kiemen, Laura D. Wood, Terry K. Morgan, Mary C. Thoma, Jennifer M. Chu, Paul T. Spellman, Lisa M. Coussens, Ellen M. Langer, Rosalie C. Sears. A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A061.

Vitamin B5 Supports Oncogenic Metabolism in MYC-Driven Breast Cancer.

Pantothetic acid is required for metabolic activity that supports MYC-driven breast tumor growth.

Mitoribosomal synthetic lethality overcomes multidrug resistance in MYC-driven neuroblastoma

Mitochondria are central for cancer responses to therapy-induced stress signals. Refractory tumors often show attenuated sensitivity to apoptotic signaling, yet clinically relevant molecular actors to target mitochondria-mediated resistance remain elusive. Here, we show that MYC-driven neuroblastoma cells rely on intact mitochondrial ribosome (mitoribosome) processivity and undergo cell death following pharmacological inhibition of mitochondrial translation, regardless of their multidrug/mitochondrial resistance and stem-like phenotypes. Mechanistically, inhibiting mitoribosomes induced the mitochondrial stress-activated integrated stress response (ISR), leading to downregulation of c-MYC/N-MYC proteins prior to neuroblastoma cell death, which could be both rescued by the ISR inhibitor ISRIB. The ISR blocks global protein synthesis and shifted the c-MYC/N-MYC turnover toward proteasomal degradation. Comparing models of various neuroectodermal tumors and normal fibroblasts revealed overexpression of MYC proteins phosphorylated at the degradation-promoting site T58 as a factor that predetermines vulnerability of MYC-driven neuroblastoma to mitoribosome inhibition. Reducing N-MYC levels in a neuroblastoma model with tunable MYCN expression mitigated cell death induction upon inhibition of mitochondrial translation and functionally validated the propensity of neuroblastoma cells for MYC-dependent cell death in response to the mitochondrial ISR. Notably, neuroblastoma cells failed to develop significant resistance to the mitoribosomal inhibitor doxycycline over a long-term repeated (pulsed) selection. Collectively, we identify mitochondrial translation machinery as a novel synthetic lethality target for multidrug-resistant MYC-driven tumors.

An integrated cellular and molecular model of gastric neuroendocrine cancer evolution highlights therapeutic targets.

Gastric neuroendocrine carcinomas (G-NEC) are aggressive malignancies with poorly understood biology and a lack of disease models. Here, we use genome sequencing to characterize the genomic landscapes of human G-NEC and its histologic variants. We identify global and subtype-specific alterations and expose hitherto unappreciated gains of MYC family members in a large part of cases. Genetic engineering and lineage tracing in mice delineate a model of G-NEC evolution, which defines MYC as a critical driver and positions the cancer cell of origin to the neuroendocrine compartment. MYC-driven tumors have pronounced metastatic competence and display defined signaling addictions, as revealed by large-scale genetic and pharmacologic screening of cell lines and organoid resources. We create global maps of G-NEC dependencies, highlight critical vulnerabilities, and validate therapeutic targets, including candidates for clinical drug repurposing. Our study gives comprehensive insights into G-NEC biology.

Rewired Metabolism Caused by the Oncogenic Deregulation of MYC as an Attractive Therapeutic Target in Cancers.

MYC is one of the most deregulated oncogenes on multiple levels in cancer. As a node transcription factor, MYC plays a diverse regulatory role in many cellular processes, including cell cycle and metabolism, both in physiological and pathological conditions. The relentless growth and proliferation of tumor cells lead to an insatiable demand for energy and nutrients, which requires the rewiring of cellular metabolism. As MYC can orchestrate all aspects of cellular metabolism, its altered regulation plays a central role in these processes, such as the Warburg effect, and is a well-established hallmark of cancer development. However, our current knowledge of MYC suggests that its spatial- and concentration-dependent contribution to tumorigenesis depends more on changes in the global or relative expression of target genes. As the direct targeting of MYC is proven to be challenging due to its relatively high toxicity, understanding its underlying regulatory mechanisms is essential for the development of tumor-selective targeted therapies. The aim of this review is to comprehensively summarize the diverse forms of MYC oncogenic deregulation, including DNA-, transcriptional- and post-translational level alterations, and their consequences for cellular metabolism. Furthermore, we also review the currently available and potentially attractive therapeutic options that exploit the vulnerability arising from the metabolic rearrangement of MYC-driven tumors.

MDB-32. TRANSLATION OF NON-CANONICAL OPEN READING FRAMES AS A CANCER CELL SURVIVAL MECHANISM IN CHILDHOOD MEDULLOBLASTOMA

Abstract High-risk medulloblastoma is one of the most recalcitrant pediatric cancers, and children with MYC-amplified disease frequently succumb to relapsed disease. Extensive analyses of the coding genome in this disease have characterized additional somatic events in some subsets of patients, though most tumors lack targetable mutations and do not yield insights regarding their aggressive behavior. At the same time, medulloblastoma is known to exhibit extensive rewiring of translational control in MYC-driven tumors, consistent with recent genetic evidence that the impact of this transcription factor on control of mRNA translation may be the most critical aspect of its function during tumorigenesis. Therefore, to propose previously unknown mechanisms for this disease, we have investigated the functional impact of translation of non-canonical open reading frames (ORFs) across medulloblastoma model systems. We demonstrate that these ORFs are commonly translated in medulloblastoma model systems and patient tumors, correlating with disease subtype. Using genome-wide CRISPR/Cas9 screens, we found that ORFs are frequently essential for cell survival in medulloblastoma and describe widespread reliance on upstream open reading frames (uORFs) in particular. From these, we identify a uORF in the ASNSD1 gene that is selectively upregulated and required for maintenance of cell survival by coordinating the function of the prefoldin-like complex, a poorly understood complex implicated in post-translational control. Together, our findings provide a blueprint for oncogenic uORFs as critical disease mediators both in medulloblastoma and, by extension, human cancers more broadly.

IMMU-07. TACEDINALINE (CI994), A CLASS I HDAC INHIBITOR, TARGETS INTRINSIC TUMOR GROWTH AND LEPTOMENINGEAL DISSEMINATION IN MYC-DRIVEN MEDULLOBLASTOMA WHILE MAKING THEM SUSCEPTIBLE TO ANTI-CD47 INDUCED MACROPHAGE PHAGOCYTOSIS VIA NF-KB-TGM2 DRIVEN TUMOR INFLAMMATION

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified medulloblastoma, which sensitizes them to macrophage-mediated phagocytosis upon targeting the CD47-SIRPa innate checkpoint pathway. METHODS We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), medulloblastoma (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven medulloblastoma. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven medulloblastoma, with little to no activity in non-MYC-driven medulloblastoma, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting Class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in-vitro phagocytosis assays and in-vivo orthotopic xenograft models. RESULTS CI-994 displayed anti-tumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven medulloblastoma. Furthermore, RNA sequencing revealed NFκB pathway induction as a response to CI-994 treatment, followed by TGM2 expression, which enhanced inflammatory cytokine secretion. We further show interferon-gamma (IFN-g) release and cell surface expression of engulfment (“eat-me”) signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPa phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC-amplified medulloblastoma and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

TRLS-14. TARGETING REPLICATIVE STRESS IN BRAIN TUMORS BY COMBINING ATR AND PARP INHIBITION

Abstract Pediatric brain tumors like MYC-amplified Medulloblastomas (MB) and Atypical Teratoid and Rhabdoid Tumors (AT/RT) continue to have poor survival rates despite recent advances in understanding tumor biology. MYC is an oncogenic transcription factor often expressed in MB and AT/RT at high levels. While direct inhibition of MYC by small molecules is an active area of research, such approaches lack clinical validation and are not yet actionable. Strategies to target MYC-driven brain tumors that are immediately translatable are urgently needed. Replication stress has been identified as a prominent source of tumorigenesis over the past decade and is a potential target for cancer therapy. Specific oncogenes induce elevated levels of replicative stress through aberrant activation of transcription and dysregulation of DNA replication. In these instances, replicative stress could be rescued by either metabolic supplementation or MYC expression. The activation of ATR and PARP are critical events in response to replicative stress. PARP inhibitors stall replication forks and promote the generation of single-strand DNA, which activates ATR. Genetic and pharmacological evidence suggests the sensitization of MYC-induced tumors to PARP inhibitors. Preclinical studies in diverse tumor backgrounds have demonstrated that the combination of ATR and PARP inhibition is synergistic and leads to premature entry into mitosis, enhanced replicative stress and cell death, and tumor regression. Additionally, a reduction in ATR activity can be lethal in oncogene-induced replication stress. Using high throughput drug screening across MB and AT/RT cell lines, we found that the CNS penetrant PARP inhibitor AZD9754 significantly potentiated the cytotoxicity of ATR inhibition. This emerging synergistic combination therapy reveals new therapeutic targets in MYC-driven replicative stress. Studies are ongoing to validate in vitro findings in vivo.

Abstract B035: Leveraging therapy-induced senescence for prostate cancer immunotherapy

Abstract Most prostate cancers are immunologically “cold” with poor tumor immunogenicity and a lack of cytotoxic T cells, and as such are generally unresponsive to immune checkpoint blockade (ICB) therapies that can reactivate T cell immunity and lead to durable and curative responses in other treatment-refractory solid tumors. We recently demonstrated that therapeutic induction of a durable growth arrest phenotype known as cellular senescence in the setting of pancreatic cancer can be novel approach to make “cold” tumors “hot” and sensitize them to anti-PD-1 ICB (Ruscetti et al. Cell 2020). This senescence-driven immunological effect is mediated through induction of immunomodulatory cell surface molecules, including MHC-I expression required for antigen presentation to T cells, and activation of the senescence-associated secretory phenotype (SASP), a collection of pleiotropic cytokines, chemokines, and other growth factors that can remodel the surrounding tumor-immune landscape. While this acute SASP activation can promote cytotoxic CD8+ T and Natural Killer (NK) cell anti-tumor immunity in some contexts, chronic SASP can alternatively lead to immune suppression and tumor cell invasion and metastasis that contributes to relapse after therapy. Here we set out to characterize the senescence-inducing capacity of chemotherapies, DNA repair inhibitors, and cell cycle targeting agents implicated in cellular senescence and used in the treatment of castration-resistant prostate cancer (CRPC), and the impact of their subsequent SASPs on immune responses and therapy outcomes. Using a suite of genetically-defined murine prostate cancer cell lines and an in vitro pipeline to rapidly define senescence phenotypes following treatment, we found that many commonly used cancer therapeutics can induce cellular senescence, with chemotherapies, Aurora kinase inhibitors, and CDK inhibitors producing the most robust senescence-induced cell cycle arrest across conditions. Prostate tumor cells harboring compound tumor suppressor losses such as Pten, p53, and Rb1 were more susceptible to therapeutic senescence induction. Strikingly, only a subset of therapies that induce senescence-associated growth arrest produced an inflammatory SASP and induction of activating NK cell ligands and antigen presentation and processing genes necessary for NK and T cell immunity. Moreover, whereas most SASPs induced NK cell activity in vitro, they also polarized macrophages toward a pro-tumorigenic and immune suppressive M2 phenotype that may limit their anti-tumor efficacy. CDK inhibitors produced a SASP that could activate lymphocytes without inducing suppressive myeloid populations in MYC-driven tumors, suggesting their use may be a promising approach to target prostate cancers harboring the currently “undruggable” MYC oncogene. Collectively, our studies demonstrate that induction of senescence and its non-cell autonomous arm, the SASP, could be a unique approach to treat CRPC and create new immunological dependencies that can be potentially harnessed for prostate cancer immunotherapy. Citation Format: Lin Zhou, Katherine C. Murphy, Jarin Snyder, Kelly D. DeMarco, Boyang Ma, Marcus Ruscetti. Leveraging therapy-induced senescence for prostate cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B035.

Abstract A078: Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer

Abstract The advent of immunotherapies such as immune checkpoint blockade (ICB) has greatly expanded the therapeutic options available to patients with treatment-refractory solid tumors. However, patients with castration-resistant prostate cancer (CRPC) generally remain unresponsive to ICB therapy due to an immunologically “cold” prostate tumor microenvironment (TME) with limited accessibility to cytotoxic lymphocytes such as Natural Killer (NK) and T cells that can mediate potent anti-tumor immunity. During the course of tumor evolution, CRPCs acquire many genomic alterations and copy number changes that can influence therapy outcomes. Yet the impact of such genetic alterations on the immune TME and response to ICB remains largely unknown, in part due to the lack of models to functionally study them in an immune competent setting. To this end, we previously developed a novel electroporation-based genetically engineered mouse model (EPO-GEMM) to rapidly generate genetically defined prostate tumors de novo within an intact prostate TME (Leibold*, Ruscetti* et al., Cancer Discovery 2020). Here, we applied the EPO-GEMM approach to characterize the immune landscapes of distinct genetic subtypes of CRPC and subsequently identify and target tumor intrinsic mediators of immune suppression for prostate cancer immunotherapy. Using transposon systems to overexpress the MYC oncogene and CRISPR/Cas9 vectors to inactivate tumor suppressor genes (TSGs) commonly altered in CRPC (Pten, P53, Rb1, Apc), we generated a suite of genetically distinct prostate cancer-bearing EPO-GEMMs. Histological analysis revealed genotype-specific immune infiltrates, with MYC-driven tumors generally lacking cytotoxic CD8+ T cells and harboring increased numbers of regulatory T cells (Tregs). A similar correlation was also observed between MYC levels and cytotoxic lymphocyte suppression in CRPC patient samples. In particular, tumors with compound MYC overexpression and p53 loss (MP) displayed drastic NK cell suppression. Cytokine and RNA-seq profiling of primary EPO-GEMM CRPCs, tumor-derived cell lines, and isogenic Myc-CaP cells engineered with TSG losses revealed that MP alterations led to not only inhibition of inflammatory cytokine and interferon signaling and antigen presentation/processing gene expression, but also induction of VEGF signaling and increased angiogenesis in the prostate TME. Treatment of MP tumors with a VEGFR2 antibody to block VEGF signaling significantly reduced tumor growth and increased NK and CD8+T cell infiltration and activation, indicating a phenotypic switch from a traditionally “cold” TME. Our results demonstrate that the genetic configuration of prostate cancer shapes its surrounding TME. Additionally, targeting VEGF signaling may be a mechanism to overcome ICB resistance in prostate cancer. More broadly, we believe that by understanding the tumor intrinsic mechanisms driving immune suppression, we can identify rational immunotherapy combinations for CRPC based on the genetic fingerprint of a patient’s tumor for precision medicine. Citation Format: Katherine C. Murphy, Kelly DeMarco, Yvette Lopez-Diaz, Lin Zhou, Marcus Ruscetti. Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A078.

Heterofunctional polymeric degrading chimeras (HYDRAC) for targeted protein degradation.

e15106 Background: Here we utilize a novel proteomimetic polymer platform technology which packages peptide side chains as globular structures with benificial emergent properties for the development of compounds capable of targeted protein degradation. We refer to this class of compounds as Heterofunctional polYmeric DegRAding Chimeras (HYDRACs) and demonstrate the selective degradation of the oncoprotein Myc as a proof-of-concept. Methods: HYDRACs were generated using ring-opening metathesis polymerization (ROMP), resulting in structures highly resistant to proteolysis. Heterofunctional constructs were designed incorporating secondary sequences in addition to Myc-targeting H1. The degron sequence (RRRG) was incorporated to engage endogenous cellular machinery for targeted protein degradation. Immunofluorescence/confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with use of both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1 sequence, homopolymers, monomeric peptides) controls were used to assess tumor antiproliferative effects and Myc-specific responses. Myc target engagement was assessed via immunoprecipitation assays, NMR, circular dichroism, and fluorescence polarization with protein degradation monitored by Western blot. A CRISPR knockdown library screen of all known E3 ligases and mass spec was used to identify pathways responsible for HYDRAC-induced protein degradation. Results: A library of HYDRACs with narrow polydispersity and predetermined degrees of polymerization were generated. These compounds are cell-penetrating with antiproliferative effects at micromolar IC50s. HYDRAC treatment resulted in significant decreases in Myc protein levels, which was rescued by inhibition with MG132 and MLN4924. RNAseq showed selective overexpression of Myc pathway genes. Biophysical analysis showed strong HYDRAC-Myc interactions, supported by the presence of Myc protein following pull-down of biotin-terminated HYDRACs. Identification of E3 ligases implicated in the degradation pathway using a combination of a CRISPR knock down library and mass spectrometry are ongoing. Mice bearing Myc-CaP tumors showed delayed tumor growth following treatment with Myc-degrading HYDRACs. Studies on biodistribution and pharmacokinetic profile of the compound, as well as the ability to degrade other proteins of interest using combinations of degron sequences are ongoing. Conclusions: The presented work demonstrates the feasibility of HYDRACs for targeted protein degradation of Myc, providing rationale for further studies in Myc-driven tumor models. This successful demonstration of this first-in-class compound also opens the door for the development of HYDRACs targeting other proteins of interest and incorporation of different E3 ligase recruiters for tissue or disease-specific activity.

Abstract 1673: Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors

Abstract Molecular glue degraders (MGDs) of neo-substrates that function by hijacking the CRBN E3 ligase are clinically validated as therapeutics for the treatment of hematological malignancies. Novel MGDs of GSPT1, a translation termination factor, elicit dysregulated protein translation termination and subsequent induction of TP53-independent apoptosis in acute myeloid leukemia (AML) cells through activation of the integrated stress response. In addition, dysregulation of translation termination triggered by GSPT1 degradation appears to be cytotoxic specifically to MYC-driven cancers including solid tumors with translational addiction. Phase 1 clinical trials of three novel GSPT1 MGDs have been recently launched for the treatment of AML and/or MYC-driven solid tumors (NCT02848001, NCT04336982, NCT05546268, and NCT05144334). Herein, we present novel GSPT1 MGDs and their pre-clinical results against AML and solid tumors. Our current lead GSPT1 MGDs induced rapid, potent, and durable degradation of GSPT1, resulting in strong inhibition of tumor cell growth in a variety of cell lines of MYC-driven solid tumor and blood cancer (AML and acute lymphoblastic leukemia). Moreover, in ex-vivo assays using a panel of patient-derived primary AML cells, C-10386 was highly sensitive with EC50s at the nanomolar range. Furthermore, a related analogue (C-14207) showed excellent rodent PK profiles and demonstrated dose-dependent PKPD relationship and excellent efficacy in the NCI-H1155 NSCLC xenograft model. Finally, our current lead compounds showed safer profiles in various normal cells compared to other known GSPT1 degraders. Collectively, we have identified novel GSPT1 MGDs as promising development candidates for the treatment of AML and solid tumors. Citation Format: Min Sung Joo, JaeYung Lee, Eun-Jung Kim, Dong Hyuk Ki, Hunmi Choi, Joonwoo Nam, Jaewoo Park, Keon Wook Kang, Wooseok Han. Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1673.