Tumor Suppressor Genes CDKN2A Research Articles

Neoadjuvant adebrelimab plus dalpiciclib in HPV-negative locally advanced head and neck squamous cell carcinoma: A phase II clinical trial.

TPS6129 Background: The efficacy and safety of neoadjuvant immunotherapy (NAI) for treating resectable locally advanced head and neck squamous cell carcinoma (LAHNSCC) remain uncertain, and the comprehensive protocol of NAI needs further explored. The tumor suppressor gene CDKN2A plays a pivotal role in cell cycle regulation by modulating cyclin-dependent kinase (CDK) activity. Mutations in CDKN2A lead to continuous activation of CDK4/6, resulting in uncontrolled cell proliferation and tumor growth. Approximately 35.7% of HNSCC patients exhibit CDKN2A mutations, with the incidence rising to 58% among HPV-negative LAHNSCC patients. Studies have shown that combining immunotherapy with a CDK4/6 inhibitor produces a synergistic anti-tumor effect. This study aims to assess the efficacy and safety of combining anti-PD-L1 (adebrelimab) with a CDK4/6 inhibitor (dalpiciclib) as NAI in resectable LAHNSCC patients. Methods: This open-label, single-arm, prospective phase II trial will enroll LAHNSCC patients eligible for surgery. Inclusion criteria include: being 18 to 75 years old at study entry; pathologically confirmed HPV-negative HNSCC (oral, laryngeal, hypopharyngeal, and HPV-negative oropharyngeal carcinoma), determined via p16 immunohistochemistry or HPV DNA tests; with CDKN2A mutation; resectable LAHNSCC, staged III-IVB according to the UICC/AJCC 8th edition TNM system; an ECOG Performance Status score of 0 or 1; no prior relevant anti-tumor treatments; intent for curative treatment; and adequate organ function. Key exclusion criteria are active autoimmune diseases, use of immunosuppressive drugs, or systemic corticosteroids. Eligible patients receive 3 cycles of adebrelimab (1200 mg intravenously every 3 weeks, Day 1, 22 and 43) and 2 cycles of dalpiciclib (150 mg, po, every 4 weeks, day 1-21 and 29-49) before surgery. Dose adjustments are allowed based on toxicity. Surgery will follow 2-4 weeks post-NAI, with subsequent adjuvant radiotherapy or chemoradiotherapy based on risk factors after surgery. The primary endpoints are 1-year disease-free survival (defined as the time from surgical resection to local recurrence) and major pathological response (MPR, defined as ≤10% residual viable tumor cells), with secondary endpoints focusing on safety and predictive biomarkers. Clinical trial information: NCT06199271 .

Induced Pluripotent Stem Cell-Derived Fibroblasts Efficiently Engage Senescence Pathways but Show Increased Sensitivity to Stress Inducers.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

Genome-wide screening and functional validation of methylation barriers near promoters.

CpG islands near promoters are normally unmethylated despite being surrounded by densely methylated regions. Aberrant hypermethylation of these CpG islands has been associated with the development of various human diseases. Although local genetic elements have been speculated to play a role in protecting promoters from methylation, only a limited number of methylation barriers have been identified. In this study, we conducted an integrated computational and experimental investigation of colorectal cancer methylomes. Our study revealed 610 genes with disrupted methylation barriers. Genomic sequences of these barriers shared a common 41-bp sequence motif (MB-41) that displayed homology to the chicken HS4 methylation barrier. Using the CDKN2A (P16) tumor suppressor gene promoter, we validated the protective function of MB-41 and showed that loss of such protection led to aberrant hypermethylation. Our findings highlight a novel sequence signature of cis-acting methylation barriers in the human genome that safeguard promoters from silencing.

Abstract 131: Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a childhood soft tissue sarcoma composed of skeletal myoblast-like cells that lack the capacity to differentiate despite their high expression of terminal differentiation markers like myogenin. There are two main types of RMS: fusion-positive RMS (FP-RMS), which is associated with a chromosomal translocation resulting in an oncogenic fusion protein (PAX3-FOXO1), and the more common form, fusion-negative RMS (FN-RMS) which lacks this translocation and is instead thought to be driven by numerous other oncogenes. Our lab used a Bayesian-based computational algorithm to analyze NGS sequencing data from 290 RMS samples and identified 25 potential genetic drivers of RMS. We aim to use induced pluripotent stem cells as a new model to study how these drivers, including the PAX3-FOXO1 fusion gene, influence RMS formation and progression. To this aim, we have successfully made a doxycycline inducible lentiviral vector containing the full-length sequence of human PAX3-FOXO1 which we used to ectopically express PAX3-FOXO1 in two well-characterized iPSC lines. We subcutaneously injected these iPSCs into NOD/SCID mice and treated them with doxycycline to form teratomas, non-malignant tumors containing tissue from all three germ layers. We hypothesized that PAX3-FOXO1 induction in these differentiating teratomas, may have transforming capabilities and may deregulate transcriptional programs to form FP-RMS-like tumors. We have found that PAX3-FOXO1 overexpressing teratomas grew faster and histologically look more “immature” than their control counterparts but do not seem to give rise to rhabdo-like tumors. We want to further understand what other critical factors are necessary to drive RMS formation by focusing on one of our drivers of interest EZH2, a histone methyltransferase implicated as either an oncogene or a tumor suppressor in certain forms of cancer. We have data suggesting that EZH2-dependent histone methylation activity may act as a proto-oncogene with PAX3-FOXO1 to silence the expression of key tumor suppressor genes CDKN2A and CDKN2B in RMS. To this aim, we reasoned that iPSCs differentiated into myogenic progenitors, one of the potential cells of origin of RMS, will be a novel way to study the role of oncogenes like PAX3-FOXO1 and EZH2 in RMS. We have derived myogenic progenitors from iPSCs (iPSC-MPCs) that express key myogenic factors like PAX7 and MYOD and differentiate and fuse to form mature myofibers. We plan to next explore how ectopic expression of EZH2 and/or PAX3-FOXO1 alters gene expression and histone methylation and whether these alterations can drive RMS formation in these iPSC-MPCs. Using this model system of iPSC-MPCs to drive expression of these oncogenes has the potential to offer new and exciting ways to identify the most important genes in driving cancer formation hopefully providing insight into better targets for treatment of RMS. Citation Format: Celeste Romero, Yanbin Zheng, Lin Xu, Stephen Skapek. Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 131.

Abstract B079: Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is refractory to cytostatic and immuno-oncology therapeutic strategies, leaving surgical resection of the tumor as the only treatment that can significantly prolong survival. We are therefore exploring optimized treatment strategies targeting cancer cells as well as infiltrating immune cells and the tumor microenvironment (TME) in an autochthonous mouse model for PDAC. Single focal tumors are induced through in vivo electroporation of gene constructs into the pancreas which encode mutated Kras and facilitate knock out the tumor suppressor genes P53, Cdkn2a and/or Smad4 via CRISPR/Cas9. The resulting tumors recapitulate the human disease with respect to genetics, histology and TME composition. Furthermore, this model allows the generation of tumors reflecting different PDAC subtypes, as well as the resection of primary tumors in combination with the (neo-)adjuvant regimens. Like the human disease, these tumors are refractory to chemotherapy and immune checkpoint blockade, whereas targeted therapy by means of MEK inhibitors merely leads to temporary delay of tumor outgrowth. We therefore tested the combination of MEK inhibitor treatment with different, potentially complementary modalities. We found the combination with agonist anti-CD40 antibodies to be highly effective. Prominent regressions are observed, and in combination with primary tumor resection long-term complete responses can be achieved even in a metastatic variant of the model. Apart from the direct cytostatic impact of MEKi on the Kras-transformed tumor cells, the mechanism of action of this synergistic drug combination involves enhancement of the anti-tumor T-cell response, suppression of CD4+ Tregs, and reduction of MDSCs and M2-type macrophages in favor of M1-type pro-inflammatory macrophages. A second highly effective regimen as identified in our experiments involves the combination of MEK inhibitors with the multi-kinase inhibitor regorafenib. In contrast to the aforementioned regimen, the complementary action of these drugs involves modulation of the TME by regorafenib. While the clinical translation of the MEKi/CD40 Ab regimen is hampered by toxicity issues encountered with agonist anti-CD40 Ab, the synergy as observed in the MEKi/regorafenib regiment provides a tangible path towards clinical testing. Citation Format: Aline S. Konrad, Tanja Hägele, Stefan Zens, Caroline Vent, Moritz Hamberger, Sarah Baiker, Aron Maes, Carl-Stephan Leonhardt, Oliver Strobel, Oliver Politz, Dieter Zopf, Daniel Baumann, Julian Mochayedi, Rienk Offringa. Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model [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 B079.

Abstract B177: The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers

Abstract Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene occurs in approximately 15% of all cancers due to its proximity to the commonly deleted tumor suppressor gene CDKN2A. Elevated methylthioadenosine (MTA) levels, driven by loss of MTAP, compete with the methyl donor S-adenosylmethionine (SAM) for binding to protein arginine N-methyltransferase 5 (PRMT5), placing the methyltransferase in a hypomorphic state, and vulnerable to further PRMT5 inhibition. The screening of a DNA-encoded library, designed to identify small molecules that preferentially bind to PRMT5 in the presence of MTA, resulted in the identification of AM-9934 as a promising hit. Using structure-based drug design, potency and cooperativity were enhanced to deliver AM-9747, an MTA-cooperative PRMT5 inhibitor suitable for in vivo proof-of-concept studies. Further optimization led to AMG 193, an orally bioavailable and MTA-cooperative PRMT5 inhibitor with potent biochemical and cellular activity, and an improved pharmacokinetic profile. Insights into the binding mode of AMG 193 were elucidated by the crystal structure of AMG 193 bound to the MTA-PRMT5 complex. AMG 193 activity is enriched in MTAP-null cells in vitro, as illustrated in the HCT116 isogenic pair; AMG 193 is ~40X selective for MTAP-null cells (IC50 = 0.1 mM) over MTAP WT cells (IC50 > 4 mM) in viability assays and >100X selective in an assay evaluating symmetric dimethylarginine (SDMA) levels. AMG 193 was profiled in a panel of >850 cancer cell lines, and response demonstrated preferential sensitivity in MTAP-null cells compared to WT across multiple cancer lineages, including pancreatic, lung, biliary tract, and lymphoma. Response strongly correlated with both MTAP and CDKN2A copy number loss, and cell lines that were dependent on PRMT5, as shown by RNAi and CRISPR knockdown, were most sensitive to AMG 193 treatment. In vitro mechanism-of-action studies demonstrated that PRMT5 inhibition induced DNA damage, cell cycle arrest in G2/M, and an increase in alternative mRNA splicing in MTAP-null cells. In mice oral, once-daily administration of AMG 193 selectively inhibited SDMA in tumor versus normal tissue and was well tolerated with no hematologic perturbations. Importantly, AMG 193 demonstrated robust anti-tumor activity across a broad range of MTAP-null CDX and PDX tumor models derived from NSCLC, pancreatic, and esophageal cancers. In vitro synergy was observed with the combination of AMG 193 and several chemotherapeutic agents, including carboplatin and paclitaxel, or the KRASG12C inhibitor, sotorasib. In vivo, these combinations led to enhanced anti-tumor activity relative to single agent in MTAP-null NSCLC tumor models. In February 2022, AMG 193 became the first MTA-cooperative PRMT5 inhibitor to enter clinical development and is currently being evaluated in subjects with advanced MTAP-null solid tumors in the ongoing FIH study (NCT05094336). Citation Format: Brian Belmontes, Katherine Slemmons, Siyuan Liu, Antonia Policheni, Jodi Moriguchi, Hong Tan, Fang Xie, Famke Aeffner, Matthew G Rees, Melissa M Ronan, Jennifer A Roth, Mikkel Vestergaard, Sanne Cowland, Jan Andersson, Ian Sarvary, Patricia Lopez, Nuria Tamayo, Liping H Pettus, Susmith Mukund, Jennifer R Allen, Sanne Glad, Matthew P Bourbeau. The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B177.

Updates in Management of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-associated thoracic malignancy that is usually incurable. As demonstrated in the landmark MARS2 trial, surgical resection does not improve survival outcomes and its role in managing MPM is limited. Whilst platinum-pemetrexed chemotherapy in combination with bevacizumab was the standard first-lineapproach for unresectable disease, landmark phase 3 trials have now established the role of immune checkpoint inhibitors (CPIs) in the upfront management of unresectable disease: either nivolumab-ipilimumab or carboplatin-pemetrexed-pembrolizumab. Patient selection for optimal strategy remains an ongoing question. For relapsed disease novel genomic-based therapies targeting a range of aberrations including losses of the tumour suppressor genes BAP1, CDKN2A and NF2, are being evaluated. Nonetheless, the future of MPM therapeutics holds promise. Here we overview current treatment strategies in the management of MPM.

The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality.

Approximately 15% of cancers exhibit loss of the chromosomal locus 9p21.3 - the genomic location of the tumour suppressor gene CDKN2A and the methionine salvage gene methylthioadenosine phosphorylase (MTAP). A loss of MTAP increases the pool of its substrate methylthioadenosine (MTA), which binds to and inhibits activity of protein arginine methyltransferase 5 (PRMT5). PRMT5 utilises the universal methyl donor S-adenosylmethionine (SAM) to methylate arginine residues of protein substrates and regulate their activity, notably histones to regulate transcription. Recently, targeting PRMT5, or MAT2A that impacts PRMT5 activity by producing SAM, has shown promise as a therapeutic strategy in oncology, generating synthetic lethality in MTAP-negative cancers. However, clinical development of PRMT5 and MAT2A inhibitors has been challenging and highlights the need for further understanding of the downstream mediators of drug effects. Here, we discuss the rationale and methods for targeting the MAT2A/PRMT5 axis for cancer therapy. We evaluate the current limitations in our understanding of the mechanism of MAT2A/PRMT5 inhibitors and identify the challenges that must be addressed to maximise the potential of these drugs. In addition, we review the current literature defining downstream effectors of PRMT5 activity that could determine sensitivity to MAT2A/PRMT5 inhibition and therefore present a rationale for novel combination therapies that may not rely on synthetic lethality with MTAP loss.

JS04.5.A HEMIZYGOUSCDKN2A DELETION CONFERS WORSE SURVIVAL OUTCOMES IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND The tumor suppressor gene CDKN2A is frequently deactivated in cancer. Homozygous CDKN2A deletion is a Grade 4 defining criterion for IDH-mutant gliomas. However, the prognostic value of hemizygous CDKN2A deletions is unclear. We assessed CNV profiles of 1238 initial and 479 recurrent IDHmut gliomas to evaluate the association of CDKN2A hemizygous deletions with overall survival (OS) outcomes. MATERIAL AND METHODS The GLASS primary/recurrent glioma dataset was used to infer CDKN2A status with DNAseq (n=210) and DNA methylation array (n=100) data. Validation was conducted using DFCI, MSKCC, TCGA and DKFZ cohorts. RESULTS Longitudinal analyses of IDH-mutant gliomas without 1p-/19q-codeletion showed significant increases in CDKN2A homozygous deletion from n=6 to n=23 (5% to 20%, P=0.0001, Fisher’s-exact test) and hemizygous deletion from n=19 to n=34 (17% to 30%, P=0.002) at recurrence. CDKN2A hemizygous deletions were significantly associated with poor OS at initial diagnosis (P<0.0001, log-rank test) and recurrence (P<0.0001), remaining significant in multivariable cox regression model that accounted for age, treatment with radiotherapy and/or alkylating agents, and 1p/19q-codeletion status: HR 2.40 (95%-CI: 1.31-4.37, P=0.004). Validation in independent datasets confirmed increased frequencies of CDKN2A hemizygous deletions in recurrent cases as well as the association between CDKN2A hemizygous deletion and worse OS in IDHmut gliomas. Using the DKFZ DNA methylation profiling dataset we further confirmed that CDKN2A status can be accurately assessed through DNAseq and DNA methylation array profiling. CONCLUSION CDKN2A hemizygous deletion is associated with significantly worse OS in initial and recurrent IDHmut gliomas. Similar to CDKN2A homozygous deletion, CDKN2A hemizygous deletion is enriched in post-treatment, recurrent IDHmut gliomas, confirming the value of CDKN2A status (re-)assessment in recurrences. Our results highlight the importance of incorporating CDKN2A status in diagnostic workup to inform prognosis and treatment strategies, revealing the previously unrecognized prognostic value of hemizygous CDKN2A deletions.

PhD Project: Predictive and Functional Evaluation of Patients Derived Tumor Spheroids and Organoids-Investigating the Relationship between Size and Testing Performance

PhD Project: Predictive and Functional Evaluation of Patients Derived Tumor Spheroids and Organoids-Investigating the Relationship between Size and Testing Performance

Novel strategy for oncogenic alteration-induced lipid metabolism reprogramming in pancreatic cancer.

The pathogenesis of pancreatic cancer involves substantial metabolic reprogramming, resulting in abnormal proliferation of tumor cells. This tumorigenic reprogramming is often driven by genetic mutations, such as activating mutations of the KRAS oncogene and inactivating or deletions of the tumor suppressor genes SMAD4, CDKN2A, and TP53, which play a critical role in the initiation and development of pancreatic cancer. As a normal cell gradually develops into a cancer cell, a series of signature characteristics are acquired: activation of signaling pathways that sustain proliferation; an ability to resist growth inhibitory signals and evade apoptosis; and an ability to generate new blood vessels and invade and metastasize. In addition to these features, recent research has revealed that metabolic reprogramming and immune escape are two other novel characteristics of tumor cells. The effect of the interactions between tumor and immune cells on metabolic reprogramming is a key factor determining the antitumor immunotherapy response. Lipid metabolism reprogramming, a feature of many malignancies, not only plays a role in maintaining tumor cell proliferation but also alters the tumor microenvironment by inducing the release of metabolites that in turn affect the metabolism of normal immune cells, ultimately leading to the attenuation of the antitumor immune response and resistance to immunotherapy. Pancreatic cancer has been found to have substantial lipid metabolism reprogramming, but the mechanisms remain elusive. Therefore, this review focuses on the mechanisms regulating lipid metabolism reprogramming in pancreatic cancer cells to provide new therapeutic targets and aid the development of new therapeutic strategies for pancreatic cancer.

Methylation study of tumor suppressor genes in human aberrant crypt foci, colorectal carcinomas, and normal colon.

Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls. In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed. %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation. Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.

Abstract 1563: Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas

Abstract Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) and lung cancer mainly harbor disruptive mutations in TP53 and/or CDKN2A tumor suppressor genes, which are associated with refractoriness to treatment as well as poor prognosis and survival. Mutational loss of TP53 function fosters elevated expression of Aurora kinase A (AURKA) that plays a crucial role in mitotic progression at G2/M and stabilizes DNA replication forks at S phase. We have previously demonstrated that combined inhibition of AURKA and WEE1, a G2/M checkpoint kinase induced by replication stress, results in synergistic anti-tumor effects in HNSCC in vitro and in vivo. We have now further confirmed synergy using the highly selective AURKA inhibitor VIC1911 in HNSCC and lung cancers harboring TP53 mutations and explored mechanisms of cell death. VIC1911 and adavosertib combination synergistically suppressed cell growth and survival in both 2D- and 3D-culture systems relative to vehicle or single-agent treatment in TP53-mutated HNSCC FaDu and CAL27, and lung cancer A549 and NCI-H358 cells, with no observable toxicity in normal cells, predicting a favorable therapeutic index. Furthermore, combination treatment accelerated mitotic entry and resulted in accumulation of mitotic catastrophe, as demonstrated by time-lapse imaging, and apoptotic cell death as evidenced by cleaved PARP assays. Notably, we found that combination VIC1911 and adavosertib in HNSCC cells drastically slowed and stalled progression of replication forks in DNA fiber analysis (Mean: DMSO=3.117; adavosertib=1.495; VIC1911=0.9757; combination=0.5548 kb/min; P &amp;lt;0.0001) and amplified DNA damage as measured by γH2AX, indicating induction of severe replication stress. In vivo, this combination resulted in significant tumor regression in both HNSCC and lung adenocarcinoma patient-derived and cancer cell-derived xenografted mice compared with either vehicle or single-agent treatment. Taken together, these results suggest that AURKA inhibition increases dependency on WEE1 by enhancing replication stress and mitotic catastrophe, and support clinical evaluation of combined AURKA and WEE1 inhibition as a novel and effective treatment for HNSCC and lung cancer patients with elevated AURKA expression. Citation Format: Jong Woo Lee, Sundong Kim, Sebastian Cruz-Gomez, Jackie Shi, Cindy Yang, Barbara Burtness. Concomitant inhibition of Aurora kinase A and WEE1 kinases results in synergistic tumor control and heightens DNA replication stress in head and neck and lung carcinomas [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 1563.

Calorie restriction alters the mechanisms of radiation-induced mouse thymic lymphomagenesis.

Calorie restriction (CR) suppresses not only spontaneous but also chemical- and radiation-induced carcinogenesis. Our previous study revealed that the cancer-preventive effect of CR is tissue dependent and that CR does not effectively prevent the development of thymic lymphoma (TL). We investigated the association between CR and the genomic alterations of resulting TLs to clarify the underlying resistance mechanism. TLs were obtained from previous and new experiments, in which B6C3F1 mice were exposed to radiation at 1 week of age and fed with a CR or standard (non-CR) diet from 7 weeks throughout their lifetimes. All available TLs were used for analysis of genomic DNA. In contrast to the TLs of the non-CR group, those of the CR group displayed suppression of copy-neutral loss of heterozygosity (LOH) involving relevant tumor suppressor genes (Cdkn2a, Ikzf1, Trp53, Pten), an event regarded as cell division-associated. However, CR did not affect interstitial deletions of those genes, which were observed in both groups. In addition, CR affected the mechanism of Ikzf1 inactivation in TLs: the non-CR group exhibited copy-neutral LOH with duplicated inactive alleles, whereas the CR group showed expression of dominant-negative isoforms accompanying a point mutation or an intragenic deletion. These results suggest that, even though CR reduces cell division-related genomic rearrangements by suppressing cell proliferation, tumors arise via diverse carcinogenic pathways including inactivation of tumor suppressors via interstitial deletions and other mutations. These findings provide a molecular basis for improved prevention strategies that overcome the CR resistance of lymphomagenesis.

Abstract A051: Understanding the Arf-p53 axis in PDAC suppression

Abstract Understanding the gene regulatory networks that contribute to PDAC initiation and progression is critical for improving early detection and more effectively treating this disease. Activating mutations in KRAS are often accompanied by mutations in the TP53, CDKN2A, and SMAD4 tumor suppressor genes in ~72%, 30%, and 32% of PDAC cases (Raphael et al. 2017). Understanding the consequences of deletion of individual tumor suppressor genes can delineate specific genotype-phenotype correlations that can provide new clinical insights. Using a genetically engineered mouse PDAC model based on KrasG12D expression and tumor suppressor gene inactivation through Ptf1aCreER expression in adult acinar cells, we showed previously that p53 deletion or point mutation dramatically accelerates PDAC relative to mice with intact p53. To understand tumor evolution in mice with different p53 statuses, we analyzed the transcriptomic profiles of PDACs developing with intact or mutant p53 by spatial transcriptomics and both bulk and single cell RNA-sequencing. These experiments have provided insight into the pathways that become dysregulated as PDAC develops in the context of different p53 alleles. In addition, as oncogenic signals trigger p53 stabilization via the p19ARF (ARF) protein in some contexts, we sought to determine whether ARF acts upstream of p53 in PDAC suppression. Indeed, the frequent deletion of the CDKN2A locus, which encodes both the ARF and p16INK4A (p16) tumor suppressor genes, suggests the importance of both these genes in suppressing PDAC, but the specific role of ARF in PDAC suppression has not been tested. Using mouse models to definitively interrogate the importance of ARF in suppressing PDAC, we found that Arf inactivation promotes KrasG12D-driven PDAC, and with a latency similar to p53 inactivation. Interestingly, concurrent inactivation of both p53 and Arf accelerated oncogenic KRAS-driven PDAC development relative to p53 inactivation alone, suggesting a p53-independent role for ARF in PDAC suppression. Transcriptomic profiling of tumors from these mice revealed both p53-dependent and p53-independent programs of ARF-mediated tumor suppression, which provides insight into how tumors develop in the absence of ARF. Characterization of the immune and fibroblast components of the tumor microenvironment in tumors with intact tumor suppressors, p53 deficiency, Arf deficiency and combined p53;Arf deficiency reveals genotype-dependent differences, including different CAF profiles observed in tumors of different genotypes. Ongoing analysis of human PDAC will help to further parse the roles of ARF and p16 in human pancreatic cancer development. Together, these studies will help to illuminate how p53 and Arf inactivation contribute to cancer cell evolution and crosstalk with the tumor microenvironment during PDAC development, understanding with potential for improving clinical interventions in PDAC. Citation Format: Laura D. Attardi, Brittany M. Flowers, Kathryn Hanson, Abigail S. Mulligan, Sofia Ferreira, Sohinee Bhattacharyya, Hannes Vogel, Laura D. Wood, Mara Sherman. Understanding the Arf-p53 axis in PDAC suppression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A051.

Abstract IA008: The origins and function of copy number alterations in pancreas cancer

Abstract Pancreas ductal adenocarcinoma is a devastating disease that lacks effective treatment options. Often considered genetically homogenous, it is initiated by activation of oncogenic Kras and often sustains inactivating mutations in the TP53, CDKN2A, and SMAD4 tumor suppressor genes. However, additionally, advanced full blown PDAC often has a large degree of somatic copy number alterations (CNA) whose significance to disease etiology are poorly understood. Interestingly, gene mutations and copy number events may be interconnected, as TP53 mutations are strongly associated with the emergence of tumors harboring large number of CNAs, detectable chromothripsis, extensive intratumoral heterogeneity, and are often polyploid. Using new mouse modeling and genetic engineering approachs, we have begun to explore the emergence and functionality of copy number alterations in human PDAC. The current presentation will focus on the evolution of highly rearranged tumors that arise in Kras mutant pancreatic epithelial cells that sporadically inactivate p53, as well as the cell extrinsic functions of so-called CDKN2A deletions. The work highlights the fundamental importance of copy number events in PDAC. Citation Format: Scott W. Lowe. The origins and function of copy number alterations in pancreas cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA008.

Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

EP08.02-116 Design of a Phase 1 Study of AMG 193, an MTA-Cooperative PRMT5 Inhibitor, in Patients with Advanced MTAP-Null Solid Tumors

Protein arginine methyltransferase 5 (PRMT5) is an emerging target for cancer treatment. MTAP homozygous deletion occurs in 15% of cancers and often coincides with deletion of the tumor suppressor gene CDKN2A, leading to buildup of its substrate MTA. MTA shares close structural similarity to S-adenosyl methionine (SAM), the substrate methyl donor for PRMT5. By competing with SAM, MTA partially inhibits PRMT5. Thus, MTAP-null cancers are susceptible to further PRMT5 inhibition (Kryukov Science 2016).

Abstract 1807: The discovery and preclinical characterization of the MTA cooperative PRMT5 inhibitor AM-9747

Abstract Homozygous deletion of the tumor suppressor gene CDKN2A and the neighboring MTAP gene located at chr9p21 occurs in 10-15% of human cancers. Deletion of MTAP, an enzyme in methionine and adenine salvage pathways, results in accumulation of its substrate MTA, which is structurally similar to SAM, the substrate methyl donor for the type II methyltransferase PRMT5. In MTAP deleted cells, MTA competes with SAM for binding to PRMT5, placing PRMT5 in a partially inhibited or hypormorphic state. Multiple studies using shRNAi knockdown have shown that tumor cell lines harboring MTAP deletions are vulnerable to PRMT5 inhibition. PRMT5 inhibitors that have advanced to clinical studies do not selectively target the MTA-bound form of PRMT5, and the preclinical activity of these molecules is not enriched in MTAP-deleted tumor cells lines. Moreover, the therapeutic window of these molecules is narrow, presumably due to the inhibition of PRMT5 in normal cells. We set out to identify PRMT5 inhibitors that bind cooperatively with MTA, with the goal of selectively targeting PRMT5 in MTAP-deleted tumors. A DNA encoded library screen was conducted to identify small molecules that preferentially bind to PRMT5 in the presence of MTA. The subsequent optimization of screening hits to improve potency, MTA-cooperativity, and pharmacokinetic properties led to the identification of AM-9747. The nature of the MTA cooperativity of AM-9747 was interrogated by multiple biophysical methods and structural biology experiments. Following treatment with AM-9747, the levels of SDMA marks were lower in HCT116 MTAP-deleted cells (IC50 = 0.0002 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.050 μM). AM-9747 selectively inhibited the proliferation of HCT116 MTAP-deleted cells (IC50 = 0.027 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.63 μM). The profiling of AM-9747 in an expanded panel of tumor cell lines demonstrated that AM-9747 inhibited the proliferation of most MTAP-deleted cells, with minimal effects on MTAP-WT cells. In vitro mechanism of action studies demonstrated that treatment with AM-9747 induces DNA damage, as illustrated by increased phosphorylation of H2AX, and an arrest in the G2/M phase of the cell cycle in MTAP-deleted cells. In vivo, oral administration of AM-9747 selectively inhibits SDMA and tumor growth in HCT116 MTAP-deleted tumor xenografts, compared to HCT116 MTAP-WT xenografts. Furthermore, treatment with AM-9747 inhibits the growth of multiple MTAP-deleted tumor xenograft models, including BXPC3 (PDAC) and DOHH2 (DLBCL). AM-9747 was profiled against a panel of over twenty PDX models, with greater than 50% tumor growth inhibition observed in the majority of PDX models harboring deletion of the MTAP gene. Our data with AM-9747 indicates that PRMT5 inhibitors that selectively target PRMT5 in cooperation with MTA may represent a novel and compelling therapeutic strategy for the treatment of MTAP-deleted cancers. Citation Format: Brian Belmontes, Antonia Policheni, Siyuan Liu, Katherine Slemmons, Jodi Moriguchi, Hayley Ma, Daniel Aiello, Yajing Yang, Mikkel Vestergaard, Sanne Cowland, Jan Anderson, Ian Sarvary, Nuria Tamayo, Liping Pettus, Susmith Mukund, Leszek Pope, Jennifer R. Allen, Sanne Glad, Matthew Bourbeau, Paul E. Hughes. The discovery and preclinical characterization of the MTA cooperative PRMT5 inhibitor AM-9747 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1807.

EPEN-32. Leveraging cell surface targets as therapeutic vulnerabilities for pediatric ependymoma

Brain tumors are the leading cause of cancer-associated death in children. Ependymoma, an aggressive type of childhood brain tumor, is currently treated with surgery and radiotherapy. Ependymomas are a molecularly heterogeneous group of tumors driven by distinct genetic and epigenetic alterations. In children, 90% of ependymomas arise intracranially, with two thirds occurring in the posterior fossa (PF) and one third in the supratentorial brain (ST). PF ependymomas are divided into at least two groups termed, PFA and PFB, with PFA tumors associated with poor clinical outcomes. Over 70% of ST ependymoma are characterized by an oncogenic fusion between ZFTA and RELA and shown in some cohorts to have poor clinical outcome, particularly in the context of CDKN2A tumor suppressor gene loss. A major challenge in identifying therapies against ependymoma, has been the paucity of genetic abnormalities available for targeting. PFA ependymomas harbor largely balanced genomes with no recurrent CNVs, fusions, or somatic mutations that are amenable to pharmacologic inhibition. ZFTA-RELA ependymoma while representing a clear disease driver, functions as a transcription factor and lacks clear binding surfaces available for direct inhibition using small molecules. Therefore, alternative approaches are needed to identify new targets and effective therapies in ependymoma to be evaluated in pre-clinical models. In both human ependymoma cell culture lines and PDX models, we demonstrate that a multi-omic approach is promising for cell surface target discovery, and further, focused cell surface profiling can identify lead targets that can be rapidly translated for CAR T-cell therapy.