Loss Of Tumor Suppressor Genes Research Articles

Subcellular structure, heterogeneity, and plasticity of senescent cells.

Cellular senescence is a state of permanent growth arrest. It can be triggered by telomere shortening (replicative senescence) or prematurely induced by stresses such as DNA damage, oncogene overactivation, loss of tumor suppressor genes, oxidative stress, tissue factors, and others. Advances in techniques and experimental designs have provided new evidence about the biology of senescent cells (SnCs) and their importance in human health and disease. This review aims to describe the main aspects of SnCs phenotype focusing on alterations in subcellular compartments like plasma membrane, cytoskeleton, organelles, and nuclei. We also discuss the heterogeneity, dynamics, and plasticity of SnCs' phenotype, including the SASP, and pro-survival mechanisms. We advance on the multiple layers of phenotypic heterogeneity of SnCs, such as the heterogeneity between inducers, tissues and within a population of SnCs, discussing the relevance of these aspects to human health and disease. We also raise the main challenges as well alternatives to overcome them. Ultimately, we present open questions and perspectives in understanding the phenotype of SnCs from the perspective of basic and applied questions.

Abstract 1395: Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications

Abstract Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. We performed single-cell RNA sequencing of primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. We discovered that even a small proportion (~30%) of basal-like ductal cells in primary PDAC was associated with poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression. Diverse immune cell populations, including naive SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells. The proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. Overall, our study provides a valuable resource for better understanding the roles of ITH and TME in PDAC evolution and their clinical relevance in the treatment of PDAC. Citation Format: Hyemin Kim, Hyong-oh Jeong, Se-Hoon Lee, Kwang Hyuck Lee, Kyu Taek Lee, Jong Kyun Lee, Min Woo Lee, Semin Lee, Joo Kyung Park. Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications [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 1395.

Abstract 3069: Targeting lactic acid sensing GPR132 in ccRCC

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, accounting for 70-80% of the cases. Despite the improvements in tumor therapeutics, patients with metastatic clear cell tumors still have a five-year survival rate lower than 10%. Most of the ccRCC tumors harbor loss of VHL tumor suppressor gene on chromosome 3, leading to constitutively stabilized HIF, activation of downstream pathways involved in glycolysis, angiogenesis and metastasis and a highly acidic, vascularized, and immunosuppressive tumor microenvironment (TME). Such molecular alterations lead to the buildup of lactic acid within the tumor microenvironment. Long has been considered as a byproduct of hypoxic tumor metabolism, but recent studies have demonstrated a pro-tumorigenic role of lactic acid. Therefore, elucidating the molecular mechanisms on how tumor cells take advantage of lactic acid can identify potential novel therapeutic options. To understand how ccRCC cells sense extracellular lactic acid, I explored the concept of a lactate sensor. The currently known lactate sensors are GPR81 and GPR132. By querying TCGA data, GPR81 is reduced in ccRCC tumors and does not predict patient outcome while GPR132 is elevated in ccRCC tumors compared to normal kidney and demonstrates a strong negative effect on survival. Therefore, GPR132 stands out as a potential lactate sensor in ccRCC with meaningful impact on patient survival. Previously, GPR132 has been shown to be involved in tumor associated macrophage M1 to M2 transition upon lactic acid exposure. However, GPR132 exerts a striking impact on ccRCC tumors. Depleting GPR132 significantly decreased the HIF2α level in multiple ccRCC cell lines, establishing a molecular connection between lactic acid, GPR132 and HIF2α. Furthermore, due to suppressed HIF2α signaling, GPR132 knockout led to decreased lactic acid uptake through MCT1. Such decreased lactic transporter MCT1 abrogated the elevated aggressive phenotype in ccRCC cells when exposed to lactic acid. Metabolically, GPR132 knockout led to decreased lipid deposition. In addition, such deprivation of lipid droplet is not replenished when cells are exposed to lactic acid. Furthermore, by conducting an isotype-tracing experiment, GPR132 knockout actively decreased lactic acid uptake by the tumors and limit their resources to regenerate TCA cycle intermediates and build up lipid droplets. More importantly, GPR132 knockout led to defective mitochondria activities and activation of apoptosis, both of which led to significantly suppressed ccRCC growth in vitro and in vivo. This suggested that GPR132 likely affects ccRCC fitness through multiple layers of signaling mechanisms. Overall, our study has revealed that lactate sensing GPR132 is a critical signaling pathway for ccRCC tumor development in a glycolytic feed-forward context. As a membrane bound receptor, GPR132 holds immense potential as a novel therapeutic target in ccRCC. Citation Format: Dazhi Wang. Targeting lactic acid sensing GPR132 in ccRCC [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 3069.

Abstract 6985: The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck cancer

Abstract Head and neck cancer (HNC) is the sixth common malignancy in the world and > 90% of HNC cases are squamous cell carcinoma in origin. Oral cancer is one major HNC subtype. Despite the recent advancements in cancer diagnostics and therapeutics, the 5-yr survival rate of advanced HNC patients remains <50%. Ribosomes are the cellular factories responsible for making proteins. The hyper-activation of ribosome biogenesis initiated by oncogene gain or tumor suppressor gene loss plays a critical role in cancer initiation and progression. Ribosome biogenesis is a complex and energy intensive process that involves hundreds of factors. UTP14A gene encodes a member of the uridine triphosphate 14 family and is an essential component of a large ribonucleoprotein complex bound to the U3 small nucleolar RNA. Previous studies have demonstrated that UTP14A plays a key role in the synthesis of ribosomes and 18S rRNA, and is overexpressed in several malignancies. However, the biological functions and underlying mechanisms of UTP14A in HNC remain elusive. We found not only the increase of UTP14A mRNA expression in TCGA-HNC database but also its negative impact on patient clinical outcome. UTP14A depletion significantly impaired HNC cell proliferation, migration and invasion in vitro. Ectopic overexpression had the opposite effect, suggesting an oncogenic role for UTP14A in HNC. Through gene set enrichment analysis of pairwise RNA sequencing results, several genes involved in the process of epithelial mesenchymal transition (EMT) were potentially associated with manipulated UTP14A expression. We also detected a concordant change of lipid droplet numbers and its surface marker expression in UTP14A-manipulated cells, respectively, by BODIPY staining and immunhistochemical staining. Western blot analysis validated a positive relationship of EMT markers and COX-2, a potential mediator of lipid metabolism and EMT, with altered UTP14A expression. The involvement of COX2 in UTP14A mediation was confirmed by using both pharmacological inhibition and genetic manipulation. We also identified p38 as a central downstream effector but acting upstream from COX-2 in UTP14A mediation in oral cancer cells. We believe that our study of UTP14A-mediated oncogenic signaling axis can potentially be harnessed as a novel therapeutic target against HNC although more mechanistic studies may be needed. Citation Format: Li-Wha Wu, Chen-Ni Chang, Pulak K. Biswas, Sen-Tien Tsai. The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck cancer [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 6985.

Network targeting combination therapy of synthetic lethal vulnerabilities in 9p21-deficient glioblastoma: A case report.

Patients with relapsed or progressive glioblastoma only rarely respond to salvage therapies. Nevertheless, comprehensive genomic profiling can provide insight that can identify promising approaches. Signaling pathway analyses have revealed synthetic lethal partnerships, which create the possibility of targeting vulnerabilities arising from the loss of tumor suppressor genes. For synthetic lethal vulnerabilities that are not present in normal tissues, lethal cytotoxicity against cancer cells can be achieved without the necessity of causing normal tissue toxicity. This case report describes a patient with progressive glioblastoma with homozygous deletion of chromosome 9p21. Vulnerabilities created by CDKN2A and MTAP loss were exploited with pemetrexed, bevacizumab, and candesartan to achieve a clinically meaningful remission by targeting multiple synthetic lethal nodes. Synthetic lethality can reveal the basis for exceptional responsiveness, thus extending the utility of molecular profiling and fulfilling the promise of precision medicine.

Abstract C048: Inducible activation of sgRNA libraries in tumor xenografts empowers large-scale in vivo target discovery screens

Abstract CRISPR-mediated genome editing is a powerful approach to understanding disease biology, including identifying genes essential for cancer cell proliferation, immune evasion and survival. We routinely use the technology to identify novel therapeutic targets for cancer using both in vitro and in vivo tumor models, focusing on targets that are synthetic lethal with tumor suppressor gene loss. Large-scale CRISPR screens are often conducted using in vitro cell culture systems as in vivo ‘drop-out’ screens face several bottlenecks resulting in poor sgRNA library representation and low signal to noise ratio. These inherent limitations of in vivo screens occur because only a small fraction of the injected cells contribute to xenograft tumor formation, and because of the uneven clonal expansion due to heterogenous growth conditions in the tumor microenvironment. Hence, many in vivo screens are underpowered for statistical analysis, resulting in a high rate of ‘false negatives’ or ‘false positives’ unless a large number of animals are used or the size of the sgRNA library is greatly reduced to overcome sampling noise. Here, we validate a novel in vivo screening technology, CRISPR-StAR (Stochastic Activation by Recombination), that overcomes these challenges by (i) activating the sgRNA library in established tumors and (ii) generating internally matched-pair controls for each sgRNA using molecular barcodes to capture the history of each clone within the tumor. Using this clonal information, we developed a robust computational pipeline that extracts meaningful target sgRNA-level data from individual tumors, despite the random under-representation of the larger library. Statistical (down-sampling) analysis revealed that CRISPR-StAR has a resolution of 1,000 sgRNAs per tumor which reduces the number of animals required by 7-fold to traditional approaches. Consequently, we have performed several druggable genome screens (~30,000 sgRNA) using just 30-40 individual tumors and identified a catalog of tumor suppressor genes that, when lost, strongly promote tumor growth in vivo without affecting cell proliferation in vitro. This group of genes is enriched with epigenetic modifiers, in particular multiple members of the COMPASS family and the SWI/SNF chromatin remodeling complexes. These results validate CRISPR-StAR as a powerful in vivo functional genomics platform for high throughput target discovery screens. Citation Format: Silvia Fenoglio, James Tepper, Lauren Grove, Esther CH Uijttewaal, Alborz Bejnood, Yi Yu, Hsin-Jung Wu, Annabel Devault, Shangtao Liu, Binzhang Shen, Samuel R Meier, Ashley H Choi, Tenzing Khendu, Hannah Stowe, Minjie Zhang, Brian B Haines, Alan Huang, Jannik N Andersen, Xuewen Pan, Ulrich Elling, Teng Teng. Inducible activation of sgRNA libraries in tumor xenografts empowers large-scale in vivo target discovery screens [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 C048.

Whole-Genome Sequencing Identifies Structural Variation As a Key Driver of Disease Relapse and Aggressive Clinical Behavior in Multiple Myeloma

INTRODUCTION: Many key genomic events leading to relapse in multiple myeloma (MM) occur outside of coding regions and can only be identified using whole-genome sequencing (WGS). Studies of MM progression examining single nucleotide variants (SNVs) and insertions/deletions (InDels) have identified a distinct pattern of mutations associated with resistance, however these do not fully account for drivers of progression. Studies of relapsed patients provide hints about the molecular mechanisms driving progression based on identification of homozygous loss of tumor suppressor genes, aberrant copy-number variants (CNVs), and rare examples of complex structural variants (SV). We have analyzed a set of presentation and relapsed MM samples, including paired data, derived from WGS of plasma cells and focused on SVs as key drivers of progression. METHODS: WGS was performed on 338 MM patient samples using CD138-sorted plasma cells, among which 237 samples were paired including 101 patients (30%) at presentation and at relapse, 35 of whom had more than one sample at relapse. A bioinformatics pipeline employing a consensus mechanism for determining the final set of somatic events was used based on Mutect2, Strelka2, and VarScan2 for SNVs; Mutect2, Strelka2, VarScan2, and SvABA for InDels; Battenberg and FACETS for CNVs; Manta, SvABA, DELLY2, and IgCaller for SVs. Additionally, an admixture workflow was used to estimate each individual's ancestral lineage using continentally-distinct references, comprising 23 regional populations within 5 super-populations from the 1000 Genomes Project (https://github.com/pblaney/mgp1000). Complex rearranged genomes were reconstructed using the graph-based R package JaBbA (https://github.com/mskilab-org/JaBbA). Additionally, the python package Pairtree (https://github.com/morrislab/pairtree) was used to describe the evolutionary history of acquired mutations in these patients. RESULTS: Themedian age of the cases studied was 67, and 42% were female. Racial admixture correlated with self-identification, including 74% of European ancestry with the remainder being predominantly of African ancestry. High-risk cytogenetic features were found in 10% and 13% at diagnosis and relapse, respectively. As expected, the tumor mutational burden of the relapse cases was higher than that at presentation. The patterns of SNV mutational drivers were similar at relapse in comparison to presentation with again evidence for a role of biallelic tumor suppressor gene inactivation as being a key mechanism. We also found that the number of SVs increased at relapse. Complex SVs including templated insertions, chromoplexy, and chromothripsis were detected and occurred exclusively at relapse in some cases as well as earlier in the natural history in others, suggesting they can occur as both early and late driver events. Characterizing complex SVs, further we identified them at lower levels in cases at presentation compared to relapse, consistent with these being at a higher clonal fraction and providing further evidence for their role as driver events. We did not see the emergence of additional rearrangements at relapse in either the templated insertions or chromoplexy events, suggesting that these occur at a single timepoint and remain structurally stable overtime. CONCLUSIONS: Complex SVs provide a novel mechanism driving relapse in MM which can deregulate multiple genes simultaneously providing new potential markers of aggressive disease behavior and disease evolution.

Natural Killer Cells Are Active Against Myeloma, Leukemia or Lymphoma Cells with Loss of Tumor Suppressor Genes

The therapeutic targeting of tumor cells with loss of function (LOF) for tumor suppressor genes (TSGs) is challenging across cancers, including hematologic neoplasias, because pharmacological mechanisms to restore the function(s) of such genes are not readily feasible, in contrast to e.g., inhibition of oncogenic drivers. We reasoned, however, that, although LOF for TSGs leads to de-repressed growth of neoplastic hematopoietic cells, it may not necessarily protect them from immune attack. We thus explored the hypothesis that CRISPR-based loss of function of TSGs in cells from multiple myeloma (MM), leukemias or lymphoma may still be associated with substantial response to immune effector cells such as NK cells, which have the advantage to kill tumor cells across HLA barriers. We have conducted CRISPR-based studies in 7 cell lines that represent different hematologic malignancies and different levels of sensitivity to natural killer (NK) cells, namely the B-cell lymphoma (SUDHL4), precursor B cell acute lymphoblastic leukemia (NALM6), multiple myeloma (MM1.S, LP1, KMS11), chronic myeloid leukemia (K562), and acute myeloid leukemia (MOLM14). In these genome-scale or focused CRISPR screens for LOF (CRISPR-based gene editing) or gain of function (GOF, CRISPR activation), the blood cancer lines were exposed to allogeneic donor-derived NK cells (vs. control cultures without NK cells). We evaluated the performance of genes known to represent recurrent TSGs based on genomic data of patient samples or cell lines; as well as candidate TSGs, based on results from genome-scale CRISPR gene editing screens (e.g., CERES or CHRONOS scores >0.4 in multiple DepMap releases and TPM>1 [RNA-seq]) in the same cell lines as the NK cell resistance screens. These analyses sought to identify any TSGs whose LOF may potentially alter the response of blood cancer cells to NK cells. We also evaluated genes identified as top recurrent TSGs in patient samples from MM and other hematologic neoplasias (e.g., based on prior genomic studies). On aggregate, we evaluated a collection of known and recurrent TSGs (e.g., PTEN, TP53, RB1, CDKN2C, CDKN1B, TENT5C/FAM46C) as well as other, previously underappreciated candidate genes with TSG properties in hematologic neoplasias (e.g., HIF1A, DEPDC5). Perturbation of none of these genes was identified to meet criteria for association with significant resistance to allogeneic donor-derived NK cells (e.g., log2FC>1.0, at least 3-4 sgRNAs with enrichment upon CRISPR KO or depletion with CRISPR activation, p-value <0.05, enrichment rank <100, based on rank aggregation algorithm) in any of the MM, leukemia or lymphoma cell lines examined in LOF or GOF CRISPR screens for NK cell resistance. In fact, for a limited set of cases (e.g., PTEN in KMS11 cells), KO of a TSG was associated with sensitization to NK cell treatment. To probe the mechanistic basis for this lack of effect of TSG loss, we examined the molecular sequelae of CRISPR-based KO for one of these recurrent TSGs, PTEN, by performing scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 cells expressing sgRNAs targeting select hits from our CRISPR screens and also PTEN were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis. We observed in these studies that the transcriptional changes induced in MM cells by KO of PTEN included limited, if any, changes in the expression of key genes involved in regulation of NK cell responses of tumor cells, e.g., ligands for activating or inhibitor receptors in NK cells, death receptors (e.g., TRAIL, Fas) or their downstream effectors/regulators or other molecules identified from our aforementioned genome-scale and focused CRISPR LOF and GOF studies. Notably, our in-house results from studies of NK exposure of cell lines from hematologic neoplasias are concordant with results for these TSGs in CRISPR screens of non-hematological cancer cells treated with cytotoxic T-cells (e.g., 4T1 or RENCA cells; GSE149933). Overall, our observations indicate that MM, leukemia or lymphoma cells that become deficient for diverse TSGs based on CRISPR-based gene editing are equally responsive to NK cells as their TSG-proficient counterparts. NK cell-based therapies may thus be a promising approach to target TSG-deficient hematologic malignancies for which specific pharmacological therapies are not currently available.

EXTH-63. MTA-COOPERATIVE PRMT5 INHIBITORS ARE EFFICACIOUS IN MTAP-DELETED MALIGNANT PERIPHERAL NERVE SHEATH TUMOR MODELS

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with limited treatment options and poor survival rates, necessitating the development of novel therapeutics. Approximately 25-50% of MPNST harbor loss of the enzyme methylthioadenosine phosphorylase (MTAP) due to a passenger deletion driven by loss of the proximal tumor suppressor gene, CDKN2A. PRMT5 was identified as a selective dependence in MTAP-deleted cells due to the accumulation of the substrate methylthioadenosine (MTA), which is itself an endogenous PRMT5 inhibitor. TNG908 and TNG462 are clinical stage MTA-cooperative PRMT5 inhibitors that demonstrate selectivity for MTAP-deleted cells over MTAP-intact cells of 15X and 45X, respectively. Previous reports show both molecules drive durable tumor regressions in xenograft models of various MTAP-deleted cancer histologies. Here, our objectives are to examine the activity of TNG908 and TNG462 in preclinical MPNST models. METHODS The proliferation effects of MTA-cooperative PRMT5 inhibitors, TNG908 or TNG462, and a SAM-cooperative PRMT5 inhibitor, GSK3326595, on MTAP-deleted and MTAP-intact MPNST cell lines were determined using CellTiter-Glo (CTG) assays. TNG908 and TNG462 were further profiled in two MTAP-deleted MPNST patient-derived xenograft (PDX) models, WU-356 and WU-386. RESULTS Incubation with the MTA-cooperative PRMT5 inhibitors, TNG908 and TNG462, selectively decreased the proliferation of MTAP-deleted MPNST cell lines relative to MTAP-intact MPNST cell lines. TNG908 and TNG462 drove dose-dependent antitumor activity including tumor regressions in the MTAP-deleted MPNST PDX models, WU-356 and WU-386, at well-tolerated doses. CONCLUSIONS The clinical stage MTA-cooperative PRMT5 inhibitors TNG908 (NCT05275478) and TNG462 (NCT05732831) are efficacious in MPNST models in vitro and in vivo and are therefore promising therapeutic agents for patients with MTAP-deleted MPNST.

Redox-Responsive Polymeric Nanoparticle for Nucleic Acid Delivery and Cancer Therapy: Progress, Opportunities, and Challenges.

Cancer development and progression of cancer are closely associated with the activation of oncogenes and loss of tumor suppressor genes. Nucleic acid drugs (e.g., siRNA, mRNA, and DNA) are widely used for cancer therapy due to their specific ability to regulate the expression of any cancer-associated genes. However, nucleic acid drugs are negatively charged biomacromolecules that are susceptible to serum nucleases and cannot cross cell membrane. Therefore, specific delivery tools are required to facilitate the intracellular delivery of nucleic acid drugs. In the past few decades, a variety of nanoparticles (NPs) are designed and developed for nucleic acid delivery and cancer therapy. In particular, the polymeric NPs in response to the abnormal redox status in cancer cells have garnered much more attention as their potential in redox-triggered nanostructure dissociation and rapid intracellular release of nucleic acid drugs. In this review, the important genes or signaling pathways regulating the abnormal redox status in cancer cells are briefly introduced and the recent development of redox-responsive NPs for nucleic acid delivery and cancer therapy is systemically summarized. The future development of NPs-mediated nucleic acid delivery and their challenges in clinical translation are also discussed.

Functional loss of tumor suppressor genes detected by loss of heterozygosity, but not driver mutations, predicts aggressive lymph node status in papillary thyroid carcinoma

BackgroundRecognizing aggressive tumor biology is essential to optimizing patient management for papillary thyroid carcinomas (PTC). Aggressive lymph node (ALN) status is one feature that influences decision-making. We evaluated genomic deletions in regions of tumor suppressor genes, detected by loss of heterozygosity (LOH) analysis, to understand causal alterations linked to thyroid cancer aggressiveness and to serve as a molecular diagnostic biomarker for ALN status. MethodsWe analyzed 105 primary PTC enriched for patients with ALN (64% with, 36% without). We also analyzed 39 positive lymph nodes (79% with, 21% without ALN). LOH was determined using a panel of 25 polymorphic microsatellite alleles targeting 10 genomic loci harboring common tumor suppressor genes. Additionally, ThyGeNEXT® and ThyraMIR® assays were performed. ResultsLOH was detected in 43/67 primary PTC from patients with ALN status, compared with only 5/38 primary PTC without ALN (minimal metastatic burden) (P=0.0000003). This is further supported by post hoc analyses of paired primary and metastatic samples. Paired samples from patients with ALN are more likely to harbor LOH, compared to the ALN negative group (P=0.0125). Additionally, 12/31 paired samples from patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. No association was seen between ALN and mutational, translocation, or microRNA data. ConclusionsLOH detected in primary PTC significantly predicts ALN status. Analysis of paired primary and metastatic samples from patients with / without ALN status further supports this relationship. The acquisition of LOH at additional loci is common in lymph nodes from patients with ALN status. Simple summaryA subset of patients with papillary thyroid carcinoma (PTC) will develop recurrent disease. One known predictor of recurrence is the American Thyroid Association category “Aggressive Lymph Node” (ALN) disease, considering metastatic burden. Loss of heterozygosity (LOH) — chromosomal loss in regions of tumor suppressor genes — has yet to be investigated as a possible mechanism driving ALN status in PTC. The ability to predict ALN status prior to surgery can guide the extent of surgery and postoperative treatment options. We found that paired samples from patients with ALN are more likely to harbor LOH, compared to patients without ALN disease. 38% of patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. LOH complements current molecular analysis of thyroid cancer when searching for evidence of aggressive biology.

Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD

Stem cell gene therapy using the MFGS-gp91phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

P-182 Natural killer cells are active against myeloma cells with loss of tumor suppressor genes

P-182 Natural killer cells are active against myeloma cells with loss of tumor suppressor genes

Therapeutic Targeting of DNA Replication Stress in Cancer.

This article reviews the currently used therapeutic strategies to target DNA replication stress for cancer treatment in the clinic, highlighting their effectiveness and limitations due to toxicity and drug resistance. Cancer cells experience enhanced spontaneous DNA damage due to compromised DNA replication machinery, elevated levels of reactive oxygen species, loss of tumor suppressor genes, and/or constitutive activation of oncogenes. Consequently, these cells are addicted to DNA damage response signaling pathways and repair machinery to maintain genome stability and support survival and proliferation. Chemotherapeutic drugs exploit this genetic instability by inducing additional DNA damage to overwhelm the repair system in cancer cells. However, the clinical use of DNA-damaging agents is limited by their toxicity and drug resistance often arises. To address these issues, the article discusses a potential strategy to target the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), which plays a central role in the DNA replication and damage response network. Small molecule and peptide agents that specifically target caPCNA can selectively target cancer cells without significant toxicity to normal cells or experimental animals.

PILOT PHASE II STUDY OF SELINEXOR IN COMBINATION WITH IFOSFAMIDE, ETOPOSIDE AND DEXAMETHASONE IN PATIENTS WITH RELAPSED OR REFRACTORY PERIPHERAL T‐CELL LYMPHOMAS (R/R PTCLS)

Introduction: The prognosis of R/R PTCLs is very poor. Selinexor is an oral, first in class, potent selective inhibitor of nuclear exportin, that shown promising overall response rate (ORR) and complete response (CR) in a phase I study of selinexor in combination with high-dose dexamethasone, ifosfamide, carboplatin and etoposide in R/R PTCLs, Tang et al (Haematologica 2021). These results are very preliminary and need to be confirmed; for this reason, we designed the PTCL S-IDE study (EudraCT number 2021-006229-23). Methods: S-IDE is a phase II multicenter pilot study of Selinexor in combination with Ifosfamide, Etoposide and Dexamethasone in patients with R/R PTCLs. We decided to omit carboplatin in order to reduce nausea and hematologic toxicity, in addition the activity of carboplatin in PTCL is uncertain. Key inclusion criteria are: age 18–75 years, R/R histologically confirmed PTCL (PTCL-NOS, angioimmunoblastic T cell lymphoma - AITL, anaplastic large cell ALK negative—ALK neg, T-helper follicular—TFH) after at least one course of anthracycline containing regimen chemotherapy (including or not high dose chemotherapy and stem cell transplantation - SCT), PS ECOG ≤2, adequate organ function. All patients will receive intravenous S-IDE on a 21-day cycle: Selinexor (40 mg on day 3, 5 and 7); Ifosfamide 5 g/mq on day 2; Etoposide 100 mg/mq on days 1–3; Dexamethasone 20 mg/day on days 3–7. Patient in CR at the end of the 4 courses receive a maintenance with selinexor (selinexor 60 mg weekly, until progression or unacceptable toxicity) or proceed to allo-SCT or auto-SCT according to donor availability, patient age and comorbidities. The primary endpoint is ORR at the end of 4 courses of S-IDE; the secondary endpoints are duration of response, 18-months PFS and OS, treatment related mortality, CR by histotypes, rate of patients able to undergo SCT, and adverse events. Exploratory biological analyses are ORR in subgroups based on GATA3 and TBX21 expression; loss of tumor suppressor genes on the CDKN2A/B-TP3 axis and PTEN-PI3K pathways as well as genetic gains and amplification of STAT3 and MYC; the presence of mutations described as associated with a poor prognosis, including CD28 mutations in AITL; TP63 rearrangement, loss of TP53, and loss of PRDM1 in ALK− ALCL; GATA3 , TP53, and/or CDKN2A in ALK− ALCL; and alterations in histone methyltransferase genes KMT2A, KMT2B, or KDM6A and FAT1 in PTCL-NOS; XPO1 expression; circulating tumor DNA (ctDNA). The sample size is 30 patients, with enrollment 18 months in 9 Italian centers. The sample size calculation refers to the primary endpoint; a sample size of 30 patients will allow to achieve a statistical power of more than 90% (94%) to test an increase of 30% of ORR (from 50% under null hypothesis to 80% under the alternative one) using a two-sided one sample binomial test with alpha =5%. The accrual is ongoing; at the end of February 2023, 2 patients have been enrolled. Keywords: aggressive T-cell non-Hodgkin lymphoma, combination therapies, ongoing trials Conflicts of interests pertinent to the abstract. A. Chiappella Consultant or advisory role Celgene-BMS, Gilead-Sciences, Ideogen, Janssen, Roche, SecuraBIO, Takeda Other remuneration: Lecture fees/Educational activities: Astrazeneca, Celgene-BMS, Gilead-Sciences, Incyte, Janssen-Cilag, Novartis, Roche, Takeda

Abstract ND12: TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers

Abstract STK11 loss of function mutations occur in 12-15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade in patients and preclinical models. STK11-deficient syngeneic mouse tumor models reflect this biology and are insensitive to anti-PD1 treatment. Informed by in vivo CRISPR-based screens, histone deacetylase 1 (HDAC1) was identified as a target gene, which when knocked out in tumor cells, reverses anti-PD1 resistance driven by STK11 tumor suppressor gene loss. Here, we describe the discovery and development of TNG260 as a potent and selective inhibitor of the HDAC1-containing complex CoREST. TNG260 inhibits CoREST deacetylase activity with 500-fold selectivity over the other HDAC1-containing complexes, NuRD and Sin3, and the HDAC3-containing complex NCoR. In preclinical studies, selective CoREST inhibition by TNG260 results in transcriptional reprogramming of STK11-mutant tumor cells, altering tumor cell cytokine secretion and markedly reducing recruitment of suppressive Treg cells to STK11-mutant tumors. Moreover, TNG260, in combination with anti-PD1 treatment, drives durable tumor regressions in multiple syngeneic STK11-mutant xenograft models. TNG260 has no anti-tumor efficacy in athymic mice, indicating the responses with TNG260 are mediated by T cells. Unlike previously developed pan-HDAC inhibitors, which are directly cytotoxic to cancer (and immune) cells, IND-enabling toxicology studies in rat and dog showed that TNG260 was well-tolerated at exposures predicted to be efficacious in humans, with bone marrow suppression only detectable at doses where TNG260 is no longer selective for CoREST inhibition. TNG260 clinical development will be among the first to combine the power of genetic patient selection and immunotherapy, evaluating patients with STK11 mutant cancers in a trial combining TNG260 and a checkpoint inhibitor. Citation Format: Leanne G. Ahronian, Minjie Zhang, Chengyin Min, Alice W. Tsai, Jacques Ermolieff, Patrick McCarren, Margaret Wyman, David Guerin, Ye Wang, Alborz Bejnood, Kenjie Amemiya, Brian McMillan, Nikitha Das, Preksha Shahagadkar, Brian Doyon, Andre Mignault, Colin Liang, Vassil Elitzin, Samuel R. Meier, Ashley Choi, Yi Yu, John P. Maxwell, Brian B. Haines, Jannik N. Andersen, Heather DiBenedetto, Aaron Weitzman, Alan Huang, Xinyuan Wu. TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers [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 ND12.

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

Abstract LB267: Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma

Abstract Retinoblastoma is a cancer of the infant retina primarily driven by the loss of the Rb tumor suppressor gene, which is undruggable. Here, we identified an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. SFRP2 silencing resulted in rapid production of nitric oxide, leading to p53-dependent growth arrest of retinoblastoma cells. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell surface receptor for SFRP2 in retinoblastoma cells, that CXADR functions as a "dependence receptor," transmitting a growth inhibitory signal in the absence of SFRP2, and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induced nitric oxide and profoundly inhibited retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation, and is amenable to therapeutic intervention. Citation Format: Panneerselvam Jayabal, Xiuye Ma, Kathryn M. Bondra, Yidong Chen, Peter J. Houghton, Yuzuru Shiio. Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB267.

Abstract 3956: Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene

Abstract Background. Clear cell renal cell carcinoma (ccRCC) is the most frequent type of cancer in the kidney. These tumors are highly vascular and correlate with poor prognosis. Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is a truncal event of ccRCC carcinogenesis, which yields new insights for targeted therapy. Our studies identified a small molecule, STF-62247, that is toxic to cells lacking VHL compared to RCC with a functional gene. We reported that STF-62247 blocks late stages of autophagy by targeting lysosome dynamics. Moreover, we identified lysosomal vulnerability in VHL-mutated ccRCCs, which could lead cells to death. Goal. This study aims to recognize and characterize STF-62247 potential targets. Results. Using biochemical approaches, our results indicated that STF-62247 cytotoxicity is driven by PIKfyve inhibition. Binding affinity between PIKfyve and STF-62247 is around 5 nM. Intracytoplasmic vacuoles and enlargement of endolysosomes were observed, which was prevented by adding exogenous PI(3,5)P2. Moreover, PIKfyve inhibitors such as apilimod, YM201636, Vacuolin-1, APY0201 sensitize ccRCC with a loss of VHL. Finally, genetic knockdown of PIKfyve was achieved by CRISPR/Cas9 leading VHL-inactivated cells to death. Conclusion. Altogether, our studies identified, for the first time, PIKfyve as potential target for kidney cancer cells with a loss of VHL. Citation Format: Nadia Bouhamdani, Sandra Turcotte. Identifying PIKfyve as potential target in clear cell renal cell carcinoma with a loss of the von Hippel-Lindau tumor suppressor gene. [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 3956.

Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer

BackgroundHigh-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor...