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2633 Background: Older patients (pts) with cancer are underrepresented in registrational clinical trials for immune checkpoint inhibitor (ICI) therapies. There may be relevant differences in the makeup of the tumor microenvironment (TME) and in genomic signatures of cancer in older pts. This analysis explores differences in the genomic makeup of common cancers and their TME in pts ≥ 80 years (yr) of age, compared to younger pts. Methods: Next-generation sequencing of DNA (592 gene panel, NextSeq or whole-exome sequencing, NovaSeq) and RNA (whole transcriptome sequencing, NovaSeq) was performed for non-small cell lung carcinoma (NSCLC; n = 19,891), melanoma (MEL n = 2,899), and renal cell carcinoma (RCC; n = 1,333) pt samples submitted to a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was assessed by immunohistochemistry (IHC), and high tumor mutational burden (TMB-H) was defined as ≥10 mut/Mb. Pts were stratified into age subgroups of ≥80 and < 80 yr for comparison of DNA damage response (DDR) gene alterations, gene expression profiling, and TME analysis (MCP-counter; Becht, 2016). P-values were adjusted for multiple hypotheses testing (Benjamini-Hochberg) unless noted as exploratory. Results: Pts ≥80 yr accounted for 16.0%, 19.9% and 5.3% of NSCLC, MEL and RCC pts, respectively. Compared to pts < 80 yr, NSCLC and MEL pts ≥80 yr had similar DDR gene mutation rates, while BRCA1 mutations were more common in MEL pts ≥80 yr (2.1 vs 0.8%; exploratory- p < 0.05). NSCLC ≥80 yr TMEs had increased abundance of fibroblasts (1.09-fold, p < 0.01), dendritic cells (1.07-fold, p < 0.01) and macrophages (1.04-fold, p < 0.01), and MEL≥80 yr TMEs had fewer infiltrating T-lymphocytes (0.87-fold, p = 0.02). Increased expression of immune checkpoint (IC) genes PDCDL1G2 (PD-L2; 1.11-fold), HAVCR2 (TIM-3; 1.11-fold ), and CD80/86 (1.07/1.08-fold, p < 0.05) was seen in NSCLC pts ≥80 yr, while IL-6 expression was decreased (0.88-fold; p < 0.05). The largest change in IC gene expression was for IL-6 (1.24-fold, p = 0.78) in MEL, and GZMB (0.56-fold ; p = 0.17) in RCC ≥80 yr. TMB-H was less common in NSCLC (29.7 vs 36.5%, p < 0.001) and more common in MEL pts ≥80 yr (65.7 vs 49.0%, p < 0.01), and PD-L1 (IHC-SP142, ≥2+|5%) expression was less frequent in RCC pts ≥80 yr (9.1 vs 19.4%, exploratory p < 0.05). Profiling of glutamine and glucose metabolism-related genes revealed increased SLC38A5 (1.17-fold; p < 0.0001) and decreased G6PC (0.65-fold, p < 0.01) expression in NSCLC ≥80 yr. While not statistically significant, MEL and RCC pts ≥80 yr had opposite trends for SLC38A5 and G6PC expression. Conclusions: Our analysis provides new insights to immune landscape of NSCLC, MEL, and RCC pts ≥80 yr. Differential gene expression and TME composition changes in this population suggest unique, cancer-specific therapeutic opportunities, and a potential to explore biomarkers of response to ICIs.

3589 Background: In patients with colorectal cancers (CRCs), prior studies have reported that various TP53 mutations have prognostic significance. The anatomic location of the primary CRC and the TP53 mutation subtype influence patient survival. In this study, we explored the prognostic significance of TP53 mutations (mTP53) classified as gain-of-function (GOF) or non-GOF in patients with right-sided (RCC) and left-sided CRCs (LCC). Methods: CRC specimens (6,248 RCCs and 14,215 LCCs) were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing (NGS) of DNA (592-gene panel or whole- exome sequencing). R175H, R248W, R248Q, R249S, R273H, R273L, and R282W were defined as GOF mTP53 and all other mTP53 were defined as non-GOF mTP53. MSI-H/dMMR status was determined by immunohistochemistry (IHC) of MMR proteins and/or NGS. Real-world median overall survival (mOS) was obtained from insurance claims data and calculated from tissue collection to last contact using Kaplan-Meier estimates. Results: GOF mTP53 and non-GOF mTP53 were identified in 15% and 39% respectively, in RCC and 17% and 46% respectively, in LCC. In RCC, the mOS for patients with wild-type TP53 ( wtTP53) vs. GOF mTP53 was 34 months (m) vs. 23m (p < 0.00001), and the mOS for patients with wtTP53 vs. non-GOF mTP53 was 34m vs. 27m (p < 0.001). In LCC, the mOS for patients with wtTP53 vs. GOF mTP53 was 35m vs. 33m (p = 0.056), and the mOS for patients with wtTP53 vs. non-GOF mTP53 was 35m vs. 35m (p = 0.32). The mOS for patients with non-GOF mTP53 vs. GOF mTP53 in RCC and LCC was 28m vs. 24m (p = 0.096), and 35m vs. 34m (p = 0.175), respectively. The prognostic value of GOF mTP53 and non-GOF mTP53 was further explored in relation to MSI-H/dMMR, RAS, BRAF, and PIK3CA mutation status. The worse prognosis associated with mTP53 in RCC was seen in all comparisons, except in GOF mTP53/MSI-H/dMMR, and non-GOF mTP53/ wtKRAS subgroups. In patients with LCC, worse prognosis associated with GOF mTP53 and non-GOF mTP53 was only noticeable in KRAS and PIK3CA mutant subgroups. Conclusions: This is the largest study to explore TP53 mutations and their prognostic significance in patients with RCC and LCC. The prevalence of GOF mTP53 and non-GOF mTP53 was higher in LCC compared to RCC. However, both GOF mTP53 and non-GOF mTP53 were associated with worse mOS for patients with RCC, but not LCC. Our study validates the sidedness-dependent prognostic significance of TP53 mutations. It also shows that the worse prognosis of mTP53 is independent of the approach of collectively classifying TP53 mutations into GOF vs non-GOF. Given the sheer extent and diversity of TP53 mutations, a more nuanced approach towards re-classification of GOF mTP53 is warranted. Detailed information on p53 mutations will be crucial for the interpretation of future clinical trials and for the design of novel therapeutic strategies.

2630 Background: MDM2/MDM4 are implicated in hyperprogression after immune checkpoint blockade (ICB). Our preclinical studies showed reduced T-cell killing of MDM2-amplified tumor cells that was overcome by an MDM2 antagonist or gene knockdown, and we observed additional tumor killing by T-cells with MDM2 inhibition plus anti-PD1. We hypothesized that MDM2/4 gene amplification/overexpression correlates with resistance to ICB and investigated the association of MDM2/4 alterations to overall survival (OS) following ICB across multiple solid tumors. Methods: Solid tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (NGS) were analyzed. MDM2/4 amplification (amp) was tested by NGS and determined as either amp4 (cutoff of >=4.0 copies) or amp6 (>=6.0) or amp8 (>=8.0). Real-world OS was obtained from insurance claims data and calculated from treatment start or tissue collection to last contact. Kaplan-Meier estimates were calculated for molecularly defined groups. X2/Fisher-Exact were used and significance determined as P-value adjusted for multiple comparisons (q

4571 Background: p53 target and cell cycle inhibitor CDKN1A/p21(WAF1) was initially not found to be mutated in cancer. TCGA analysis identified CDKN1A mutations are present but rare with frequencies of < 1%, but enrich in bladder cancer (̃8%). Truncating WAF1 mutations are associated with sensitivity to cisplatin and are associated with truncating Rb mutations in bladder cancer (RW+). We hypothesized RW+ bladder cancers may represent a unique subgroup with sensitivity to therapeutics. Methods: A total of 1104 urothelial tumors underwent molecular profiling at Caris Life Sciences (Phoenix, AZ) utilizing NGS of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS). Wilcoxon, Fisher’s exact were used for statistical significance (p value without and q value with multi comparison correction). Immune cell fraction (QuanTIseq) and pathway analysis (ssGSEA) were assessed by mRNA analysis. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Results: Concurrent truncating mutation (frameshift, nonsense) for RB1 and WAF1 were detected in 47 tumors (RW+, 4.25%) and tumors with wild-type status for both RB1 and WAF1 genes were classified as RW- group (54.08%). Tumors harboring only one RB1 or WAF1 mutation were excluded for further analysis. When compared to RW- group, RW+ tumors showed lower mutation rate of TP53 (54.5% vs 80.9%, q < 0.05), ARID1A (23.5% vs 38.3%, p < 0.05), and PIK3CA (18.4% vs 31.9%, p < 0.05). Interestingly, RW+ was mutually exclusive with FGFR3 mutation (18.0% vs 0%, p < 0.05). We further evaluated RNA expression of DNA repair and checkpoint arrest pathways. Notably, E2F pathway (Normalized Enrichment Scores, NES: 0.89 vs 0.86, q < 0.01) and DNA G2M checkpoint (NES: 0.89 vs 0.86, q < 0.01) were found to be the most enriched in RW+ with respect to RW- group. In addition, mRNA levels of FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 were found to be overexpressed in RW+ group (q < 0.05). RW+ tumors also displayed a distinct immunological profile: They were associated with higher PD-L1 status (63.8% vs 37.3%, q < 0.01), higher median TMB (11 mut/Mb vs 8 mut/Mb, q < 0.01) and with less frequent loss of heterozygosity for HLA-DPA1 (51.1% vs 66.7%, p < 0.05), with more high-binding-affinity neoantigen load (4.78 vs 3.89, p < 0.05) to MHC proteins, consistent with the significantly more myeloid dendritic cells in in RW+ group (0.3 vs 0.04, q < 0.001). Conclusions: Concurrent truncating mutation in RB1 and WAF1 (RW+) bladder carcinomas have fewer p53, ARID1A, and PIK3CA mutations but are enriched for E2F targets, G2/M checkpoint genes, FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 and have a distinct immunological profile. The findings suggest therapeutic strategies for RW+ bladder cancers including Chk1/Wee1, PARP inhibitors, -/+ immunotherapy that may impact on clinical outcomes.

5548 Background: Clear cell carcinomas (CCC) are rare histologies outside of the kidney and are typically less sensitive to standard treatments. Genomic alterations in chromatin remodeling pathways involving ARID1A or the intracellular PI3K-mTOR signaling pathway are found in both renal and ovarian CCC. It is unclear whether CCCs originating from different anatomic sites share a common genomic landscape. This CARIS Precision Oncology Alliance project sought to determine whether CCC of different organs shared similar genomic signatures and to identify potential pathways that could be targeted in a tumor-agnostic clinical trial. Methods: CCCs (N = 861) from multiple primary tumor sites, including kidney (30.5%), ovary (39%), endometrium (23.9%), other gynecologic sites (e.g., cervix, fallopian tube, 3.3%), and miscellaneous (non-kidney or gynecologic sites, 3.3%) were analyzed at the Caris Life Sciences Laboratory (Phoenix, AZ). Using hierarchical clustering (HC) and principal component analysis (PCA), the samples were compared across 648 total genes from five metabolic gene sets consisting of angiogenesis, glycolysis, hypoxia, oxidative phosphorylation, and fatty acid metabolism. Gene Set Enrichment Analysis (GSEA) was further conducted on the samples across fifty hallmark gene sets representing specific biologic processes and expression. Samples were also analyzed for individual genomic alterations and immune-oncology associated biomarkers. PD-L1 (SP142) expression was evaluated by immunohistochemistry (positive threshold: 2+ stain intensity and ≥ 5% tumor cells). Results: HC and PCA demonstrated that renal CCC formed distinct clusters compared to non-renal CCC. Tumors from gynecologic sites could not be separated into distinct clusters. GSEA showed that the hypoxia gene set was significantly upregulated in the renal but not in non-renal CCCs. Mutations involving TP53, ARID1A, PIK3CA were found to be the most altered genes in endometrial (62%, 26%, 31%), ovarian (13%, 55%, 48%), other gynecological sites (33%, 38%, 44%), and non-gynecologic CCC (13%, 17%, 12%) respectively. PD-L1 expression, high tumor mutational burden (≥10 mutations/Mb), and deficient mismatch repair/microsatellite instability rates across sites were: kidney (11%, 2%, 2%), endometrium (13%, 12%, 7%), ovary (9%, 4%, 3%), other gynecological sites (31%, 11%, 11%), and miscellaneous sites (11%, 19%, 4%). Conclusions: Initial metabolic gene expression clustering analysis shows that CCCs do not separate by organ of origin beyond renal versus extra-renal. TP53, ARID1A, and PIK3CA were the most frequently altered genes in non-renal CCC. Out of fifty hallmark gene sets, only two were statistically significantly different among gynecological CCCs. This similarity between gynecological CCC can be leveraged by targeting pathways such as PI3K-AKT-mTOR, DNA repair, and MYC targets in a site agnostic manner. Furthermore, high PD-L1 expression is found in other gynecological sites.

4142 Background: The composition of the tumor microenvironment (TME) in PDACs is more heavily driven by mutant (mt) KRAS than any other cancer. How genomic alterations of PDAC including KRAS status affect the immune cell (IC) landscape remains unclear. Thus, we characterized IC types and the prevalence of immuno-oncologic (IO) biomarkers in PDAC by genomic and transcriptomic analysis, and investigated associations of mt KRAS with IC estimates in the TME. Our findings were compared to our previous study in CRC. Methods: A total of 4,142 PDAC and 3,727 CRC with KRAS- mts were analyzed using next-generation DNA sequencing (NextSeq, 592 gene panel or NovaSeq, WES), IHC, and whole transcriptome RNA sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by FA, IHC and NGS. TMB-H was classified based on a cut-off of >10 mutations per MB. ICs were estimated by QuantiSeq (Finotello 2019, Genome Medicine) or MCP counter (Betcht 2016, Genome Biology). Significance was determined by X2 and Fisher-Exact and p-adjusted for multiple comparisons (q+ & CD8+ T cells, T-reg, NK, myeloid dendritic and endothelial cells compared to KRAS wt. In CRC, a similar pattern was observed but more pronounced in PDAC. Immune-regulatory markers, were among multiple genes downregulated in KRAS-mt PDAC, including CTLA-4 and LAG3. Overall changes were most pronounced in cases harboring KRAS G12D, G12V, Q61, and rare KRAS variants. Conclusions: The TME of KRAS mt PDAC shows IC patterns similar to KRAS mt CRC. Actionable IO-targets, such as PDL1, are enriched in tumors harboring specific variants of KRAS mt PDAC including the targetable G12C variant. If G12D becomes druggable, it could be targetable in 35% patients with PDAC or 15% in CRC. These results demonstrate that the TME of PDAC and CRC shows immune-cold features. Tailored immunotherapeutic strategies would have to overcome these barriers in KRAS mt PDAC and CRC, possibly in combination with molecularly targeted treatment strategies.

3132 Background: Reversion mutations (RM) in homologous recombination pathway genes including BRCA1/2 have been identified in patients with ovarian, breast, and prostate cancers whose tumors have become refractory to platinum chemotherapy or PARP inhibition. Utilizing a multi-institutional molecular database, we report the prevalence of BRCA1/2 RM in a large cohort across various tumor types. Methods: Primary and/or metastatic tumor samples underwent DNA (underwent NextSeq, 592 genes; NovaSeq, whole-exome) and RNA (NovaSeq, whole transcriptome) sequencing (Caris Life Sciences, Phoenix, AZ). RM were identified by a board-certified molecular geneticist and called only if the patient had been treated with a PARPi or a platinum agent. Baseline clinical and outcomes data were obtained through linked insurance claims data. Results: Among 118,000 solid tumors profiled, RM were observed in 54 tumors samples. RMs were seen most commonly in ovarian cancer (OC), 1.5% (23/1500) of tumors with BRCA1/2 pathogenic mutations (mut), followed by breast cancer (BC) (2.4%, 17/700), endometrial cancer (1.0%, 4/400), pancreatic cancer (1.0%, 2/210), cholangiocarcinoma (2.5%, 2/80), prostate cancer (1.3%, 3/230), cervical cancer (1.4%, 1/70), cancer of unknown primary (1.0%, 1/100), and a neuroendocrine tumor of prostate (1 RM of 9 BRCA mut). Among all RM, we detected 17 in BRCA1 and 6 in BRCA2 in OC. In BC, we identified 7 RM in BRCA1 and 10 in BRCA2. Frameshift mut that restored the reading frame in BRCA1/2 were the most common type of RM. Molecular profiles of 14 high-grade serous ovarian cancers (HGSOC) with RM were compared to 87 control HGSOC with pathogenic BRCA1/2 mut without RM. Tumors with RM had lower ER expression (25% vs. 64%, p = 0.024) and higher KDM6A mut rate (15% vs. 0, p = 0.016). Additionally, TP53 mut rates were similar in RM and control (100% vs. 95%), seen in HGSOC. In patients with RM, 7 of the 14 (50%) TP53 mut were gain-of-function (GOF) while only 19 of 84 (23%) TP53 mut in the control group were GOF (p = 0.048). More detailed clinical data were available for 29 patients with RM (17 BRCA1 & 12 BRCA2). Among these patients, 7 had received prior platinum-based chemotherapy (carboplatin or cisplatin), 7 patients were treated with PARP inhibitors (olaparib or rucaparib), or both (n = 7). Notably, 5 patients had been treated with carboplatin (n = 2, ovarian), olaparib (n = 1, breast), or both agents (n = 2, ovarian and prostate) after the detection of RM. Conclusions: This dataset is one of the largest reporting on the prevalence of BRCA1/2 RM across various tumor types. We demonstrate that the rate of RM was low among BRCA1/2 mutated tumors; this may be because some patients may not have repeat profiling post-treatment. Repeating tumor profiling at times of treatment resistance can help inform therapy selection in the refractory disease setting.

As COVID-19 continues to pose major risk for vulnerable populations, including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins, either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate the blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced the inhibition of pseudovirus infection by GLPG-0187. Because integrins activate transforming growth factor beta (TGF-β) signaling, we compared the plasma levels of active and total TGF-β in COVID-19+ patients. The plasma TGF-β1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-β1 levels correlated with activated TGF-β1 levels. Moreover, the inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and it may mitigate COVID-19 severity through decreased TGF-β1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Although the range of immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is variable, cytokine storm is observed in a subset of symptomatic individuals. To further understand the disease pathogenesis and, consequently, to develop an additional tool for clinicians to evaluate patients for presumptive intervention, we sought to compare plasma cytokine levels between a range of donor and patient samples grouped by a COVID-19 Severity Score (CSS) based on the need for hospitalization and oxygen requirement. Here we utilize a mutual information algorithm that classifies the information gain for CSS prediction provided by cytokine expression levels and clinical variables. Using this methodology, we found that a small number of clinical and cytokine expression variables are predictive of presenting COVID-19 disease severity, raising questions about the mechanism by which COVID-19 creates severe illness. The variables that were the most predictive of CSS included clinical variables such as age and abnormal chest x-ray as well as cytokines such as macrophage colony-stimulating factor, interferon-inducible protein 10, and interleukin-1 receptor antagonist. Our results suggest that SARS-CoV-2 infection causes a plethora of changes in cytokine profiles and that particularly in severely ill patients, these changes are consistent with the presence of macrophage activation syndrome and could furthermore be used as a biomarker to predict disease severity.

Objectives First-line regimens in the treatment of metastatic colorectal cancer (mCRC) combine a fluoropyrimidine with oxaliplatin (FOLFOX/XELOX) or irinotecan (FOLFIRI). There is limited efficacy data to guide the selection of one treatment over the other. This study investigated whether mutations affecting DNA damage response (DDR) could differentially influence the response to oxaliplatin and irinotecan-containing regimens. Methods We retrospectively analyzed 49 patients with mCRC for whom treatment outcomes and results of comprehensive genomic profiling of tumors were available. Specimens with at least 1 pathogenic mutation involving BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, or RAD54L were classified as DDR-mutated, while those without mutations were DDR-wild-type (WT). We compared the overall survival (OS), disease control rate, and response rate (RR) between the DDR-mutated and DDR-WT groups. Results DDR mutations occurred in 11 patients (22%). First-line treatment with an oxaliplatin-containing regimen was administered to 33 patients (31 FOLFOX, 2 XELOX), while 16 patients received FOLFIRI. Among DDR-mutated cases, first-line treatment with FOLFOX/XELOX correlated with a statistically significant improvement in median OS compared with FOLFIRI (3.4 vs.1.8 y; P=0.042) and numerically higher RR (50% vs. 33%; P=0.58). No significant difference in OS (2.4 vs. 2.5 y; P=0.42), RR, disease control rate was observed between the 2 regimens in patients with DDR-WT tumors. Conclusions Mutations in DDR genes were present in 22% of patients with mCRC. In patients with DDR-mutated tumors, initial treatment with FOLFOX/XELOX correlated with improved OS and a numerically higher RR compared with FOLFIRI.

ONC212 is a fluorinated imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo. Thus, ONC212 is a novel mitocan targeting oxidative metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

ONC201 is a promising anti-cancer agent that kills tumor cells by triggering an integrated stress response (ISR) dependent on ATF4. ONC201 demonstrated tumor regression and prolonged disease stability in patients with histone H3K27M-mutated midline glioma. The Enhancer of Zeste Homolog 2 (EZH2), a subunit of the polycomb repressive complex 2 (PRC2), is a histone methyltransferase that tri-methylates H3K27 (H3K27me3) and silences target genes. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. Based on the fact that the H3K27 mutation reduces H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of H3K27M-mutation and by increasing H3K27 acetylation with histone deacetylase inhibitors (HDACi). We evaluated synergy of EZH2i EPZ-6438 or HDACi vorinostat with ONC201 against GBM cell lines, U251 and T98G-1 and DMG cell line, SF8638. Cell viability was determined with the Cell Titer Glo assay. Apoptosis was evaluated through immunoblotting of cleaved PARP and flow cytometry analysis of cell distribution. ISR activity was evaluated using immunoblotting of ATF4. Our result demonstrate that ONC201 synergistically reduced cell viability with vorinostat in U251, T98G-1 and SF8628 cell lines, induced apoptosis in combination with vorinostat in U251 and SF8628. ONC201 synergistically reduced cell viability and induced apoptosis with EPZ-6438 in U251. The immunoblotting detected no enhancement of ATF4 by addition of EPZ-6438 to ONC201. Immunoblotting analysis showed that EPZ-6438 reduced H3K27me3 in U251. Our results unravel potent synergy between ONC201 and EZH2i or HDACi in GBM and DMG cell lines, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity.

A long-term goal in the cancer-field has been to develop strategies for treating p53-mutated tumors. A novel small-molecule, PG3-Oc, restores p53 pathway-signaling in tumor cells with mutant-p53, independently of p53/p73. PG3-Oc partially upregulates the p53-transcriptome (13.7% of public p53 target-gene dataset; 15.2% of in-house dataset) and p53-proteome (18%, HT29; 16%, HCT116-p53-/-). Bioinformatic analysis indicates critical p53-effectors of growth-arrest (p21), apoptosis (PUMA, DR5, Noxa), autophagy (DRAM1), and metastasis-suppression (NDRG1) are induced by PG3-Oc. ERK1/2- and CDK9-kinases are required to upregulate ATF4 by PG3-Oc which restores p53 transcriptomic-targets in cells without functional-p53. PG3-Oc represses MYC (ATF4-independent), and upregulates PUMA (ATF4-dependent) in mediating cell death. With largely nonoverlapping transcriptomes, induced-ATF4 restores p53 transcriptomic targets in drug-treated cells including functionally important mediators such as PUMA and DR5. Our results demonstrate novel p53-independent drug-induced molecular reprogramming involving ERK1/2, CDK9, and ATF4 to restore upregulation of p53 effector genes required for cell death and tumor suppression.

Most cancers harbor intra-tumoral hypoxia which promotes tumor progression and therapy resistance. Hypoxia-inducible factor 1α (HIF1α) mediates an adaptive response to hypoxia and contributes to multiple cancer hallmarks. We describe cancer therapeutic targeting of HIF1α by combination of CDK4/6 inhibitors (CDK4/6i) and heat-shock protein 90 inhibitors (HSP90i). CDK1 contributes to HSP90-mediated HIF1α stabilization whereas CDK1-knockdown enhances HIF1α reduction by HSP90i. Dual CDK1- and HSP90-inhibition increases apoptosis and synergistically inhibits cancer cell viability. To translate our findings, we use FDA-approved CDK4/6i in combination with HSP90i to reduce HIF1α expression and suppress viability of multiple cancer cell types, including Rb-deficient cancer cells. Overexpression of HIF1α668Epartially rescues the cell viability inhibition by combination CDK4/6i and HSP90i treatment under hypoxia. CDK4/6i and HSP90i suppresses tumor growthin vivo. Thus, combined targeting of CDK4/6 and HSP90, through a drug class effect, inhibits HIF1α and shows preclinical anti-cancer therapeutic efficacy, including with Rb-deficiency.

ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. The compound is currently being tested in patients with hematological malignancies and solid tumors, including those of the breast. We investigated strategies to convert the response of breast cancers to ONC201 from anti-proliferative to apoptotic. ONC201 treatment upregulates TRAIL and primes TRAIL-resistant non-triple negative breast cancer (TNBC) cells to undergo cell death through the extrinsic pathway. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converts the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic in a death receptor 5 (DR5)-dependent manner in vitro. Importantly, normal fibroblasts do not undergo apoptosis following rhTRAIL plus ONC201. In vivo, MDA-MB-361 tumor growth rate is significantly reduced following treatment with a combination of ONC201 and rhTRAIL as compared to control tumors. Natural killer (NK) cells which use TRAIL to kill DR5-expressing cancer cells, exhibit greater cytotoxicity against ONC201-treated breast cancer cells compared to controls. rhTRAIL also converts the response of cells from other tumor types to ONC201 from anti-proliferative to apoptotic. A monoclonal DR5-agonistic antibody converts the response of non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Our findings describe a novel therapeutic strategy that potently converts the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. This approach may be clinically relevant and has potential to induce tumor regression of patient tumors with relative resistance to ONC201 monotherapy.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19- (+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M- CSF, IL-1a, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.

Introduction Poly-(ADP)-ribose polymerase (PARP) inhibitors are successfully used for treatment of BRCA-mutated (mut) breast cancers and are under extensive evaluation for BRCA- and PALB2-mutated pancreatic ductal adenocarcinoma (PDAC). However, the optimal treatment regimen for BRCA/PALB2-mutated PDCA has yet to be established. Moreover, limited data are available on the association of BRCA/PALB2 gene alterations with other comutations and immunological biomarkers. Material and methods Tumour samples of 2818 patients with PDAC were analysed for BRCA1/2 PALB2 mutations and other genes by next-generation sequencing (NGS) (MiSeq on 47 genes, NextSeq on 592 genes). TMB was calculated based on somatic non-synonymous missense mutations. MSI-H/dMMR was evaluated by NGS, and PD-L1 expression was determined using immunohistochemistry. Results In 4.2% (n=124) of all PDAC samples BRCA mutations have been detected. BRCA2 mutations were more commonly observed than BRCA1 mutations (3.1%(n=89) vs 1.1% [n=35], p1.0% (BRCA: 21.8% vs wt 11.2%, p

Oncogenic mouse double minute 2 homolog (MDM2) is an E3-ubiquitin ligase that facilitates proteasomal degradation of p53. MDM2 amplification occurs in cancer and has been implicated in accelerated tumor growth, known as hyper-progression, following immune-checkpoint therapy. MDM2 amplification also predicts poor response to immune-checkpoint inhibitors. We sought to evaluate the role of MDM2 in T-cell-mediated immune resistance. Ovarian clear cell carcinoma cell lines carrying wild-type p53 with low/high MDM2 expression were investigated in a T-cell co-culture system evaluating T-cell-mediated tumor killing. Targeting of MDM2 was achieved by siRNA transfection or a selective MDM2 inhibitor, AMG-232 and tumor cells were tested in the T-cell co-culture system. AMG-232 activated p53 signaling in cancer cells and relative resistance to AMG-232 was observed in high MDM2-expressing cell lines. Cell lines with high MDM2 expression were more resistant to T cell-mediated tumor killing. Targeting MDM2 by gene-silencing or pharmacological blockade with AMG-232 enhanced T-cell killing of cancer cells. AMG-232 potentiated tumor cell killing by T-cells in combination with anti-PD-1 antibody treatment, regardless of changes in PD-L1 expression. The AMG-232 was not toxic to the T-cells. MDM2 inhibition lowered expression of Interleukin-6, a pro-inflammatory pro-tumorigenic cytokine. Our data support targeting MDM2 in tumors with overexpression or amplification of MDM2 as a precision therapy approach to overcome drug resistance including hyper-progression in the context of immune checkpoint therapy.

128 Background: mCRPC with alterations in genes associated with homologous recombination (HR) DNA repair (e.g., BRCA1/2) is sensitive to poly ADP-ribose polymerase inhibitors (PARPi). Preclinical studies showed that PI3K inhibitors (PI3Ki) impairs HR and sensitize cancer cells to PARPi even in the absence of HR gene mutations. These results support our hypothesis that dual PI3K and PARP inhibition may improve clinical outcomes in progressive mCRPC. We describe preliminary results of a phase Ib/II study investigating safety of the combination of copanlisib (pan-class I PI3Ki) and rucaparib (PARP-1, -2 and -3 inhibitor). Methods: Enrollment criteria included progressive mCRPC, prior androgen inhibitors (abiraterone, enzalutamide, and/or apalutamide); prior taxane chemotherapy was allowed. HR-deficiency was not required for the phase Ib. The phase I followed a standard 3+3 escalation design. Dose schema: rucaparib (continuous oral administration twice daily) 400mg (dose level [DL] -1, 1), 500mg (DL 2) or 600mg (DL 3,4) and intravenous copanlisib (45mg D1, D15 (DL -1, -2); 45mg, D1, D8, D15 (DL 1, 2, 3); 60mg, D1, D8, D15 (DL 4); 28-day cycle). Adverse events (AE) were graded by CTCAE v5.0. The primary aim of the phase I was to establish the MTD and the recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Results: Eleven pts were enrolled with a median age of 63 (55-78) and median PSA of 12 ng/mL (0.018–2,101). Seven pts (63%) received prior chemotherapy (docetaxel [7], cabazitaxel [3]). Pathogenic HR mutations included BRCA1 (1), BRCA2 (3), CDK12 (1), and FANCA (1). Treatment-related AE included grade 2 (G2) leukopenia (30%), G2 anemia (20%), G2 rash (20%). Two dose-limiting toxicities (DLTs) were observed in DL 1: G3 rash and G3 AST/ALT elevation attributed to both drugs. Six pts were treated at DL -1 without DLTs. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle). There were 2 confirmed PSA50 responses among 7 evaluable pts (28%). One pt had BRCA2 loss and 1 had PALB2 VUS (ongoing PSA response for 14 mo). Three stable disease and 1 partial response were observed among 6 pts evaluable by RECIST 1.1. Conclusions: The combination of rucaparib and copanlisib is well tolerated. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle) with signal of efficacy. Enrollment in a phase 2 expansion cohort in HR-mutated mCRPC is ongoing. Clinical trial information: NCT04253262.

Introduction: Breast cancer metastases (BCM), which cause most breast cancer (BC)-associated mortality, have increased genetic complexity compared to early-stage disease. However, the contribution of genetic alterations to site of BCM is not well-understood. Different breast cancer subtypes have varying patterns of BCM, e.g., lobular carcinoma more frequently spreads to gynecologic (Gyn) organs and the GI tract, perhaps hinting at selection pressures wherein some organs are hospitable to tumors with certain genetic alterations. Methods: Relationships between BCM site and mutations detected by DNA next-generation sequencing (NGS; NextSeq 592 gene panels or NovaSeq whole exome sequencing) were investigated using 12,464 BC samples sequenced at Caris Life Sciences (sample sizes, Table 1). PD-L1 expression was tested through IHC (Clone SP-142 (cut-off ≥1, 1%)). Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (high ≥ 10 mutations per MB). Immune cell fractions were calculated by deconvolution of whole-transcriptome data (NovaSeq) using Quantiseq (reference). Statistical significance was determined using chi-square and Wilcoxon rank sum tests adjusted for multiple comparisons. Results: Compared to primary breast tumors, BCM had increased frequency of TMB-H (10.08% vs. 4.94%), decreased PD-L1 positivity (21.09% vs. 35.82%), and were enriched for PIK3CA (34.62% vs 30.53%) and ESR1 mutations (13.34% vs 2.17%) (all P Table 1.Breast Cancer TumorsTumor SiteTotalPredominant Breast Cancer SubtypeAll12464HR+/HER2- (51.6%)Breast5014HR+/HER2- (46.5%)Liver2003HR+/HER2- (63.3%)Bone1132HR+/HER2- (69.4%)Axilla1051HR+/HER2- (47.7%)Lung823HR+/HER2- (46.1%)Lymph Node647HR+/HER2- (43.4%)Chest/Chest Wall375HR+/HER2- (44.8%)Brain359TNBC (38.2%)Other315HR+/HER2- (57.1%)Skin282HR+/HER2- (50.7%)Connective Tissue193HR+/HER2- (51.8%)GI Organs143HR+/HER2- (69.9%)Gynecologic Organs127HR+/HER2- (76.4%) Citation Format: Anna D Louie, Rani Chudasama, Sharon Wu, Marzia Capelletti, Daniel Magee, W. Michael Korn, Virginia Kaklamani, Antoinette R Tan, Pavani Chalasani, Wafik S El-Deiry, Don Dizon, Stephanie L. Graff. Mutational landscape and immune infiltration of breast cancer metastases to gynecologic and other organs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-04.

477 Background: HCC is increasingly prevalent in older adults with rising incidence and an aging population worldwide. Retrospective studies show older patients with HCC may have an increased survival compared to younger patients. However, data is lacking regarding the genomic and biologic differences, that if identified, would potentially change how we treat this disease in younger vs. older patients. Hence, there is a need to better characterize the molecular landscape of the disease in an age-specific manner. We analyzed the association of age with genomic alterations and therapeutic response to sorafenib in a cohort of advanced HCC that had undergone comprehensive molecular profiling. Methods: 487 HCC samples (excluding variants) were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Whole Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). PD-L1 positivity was determined by IHC (SP-142 clone, cutoff ≥1, 1%). Tumor mutational burden (TMB) was a measure of total somatic mutations per Mb. Immune cell populations were determined by Microenvironment Cell Population (MCP) counter analysis of RNA expression data. Overall survival (OS) calculated from tissue collection to last contact and time on treatment (TOT) with sorafenib were extracted from insurance claims and calculated using Kaplan-Meier curves. Statistical analysis was done using Chi-square, Fisher Exact and Wilcoxon rank sum tests, with p values adjusted for multiple comparisons and q77: A3 (n=75)). With age, mutational frequencies in CTNNB1 (A1=13.04%, A2=33.43%, A3=38.24%) and TERT (A1=25%, A2=68.84%, A3=76.92%) increased, while ATM (A1=6.52%, A2=0.93%, A3=1.49%) decreased (p0.05). There were fold increases in median TMB (A2/A1=1.33, A3/A1=1.33, p65 (p=0.013). Conclusions: Increased alterations in oncogenic drivers and estimates of CD8+ T cells and B cells were observed in the elderly population with HCC. The enhanced presence of co-inhibitory molecules suggests potential immune evasion. While we observed reduced TOT with sorafenib, additional studies are needed to elucidate the impact of molecular alterations on outcomes with sorafenib and newer therapies (i.e. immunotherapy) in older adults.

Plk2 is a target of p53. Our previous studies demonstrated that with wild-type p53, Plk2 impacts mTOR signaling in the same manner as TSC1, and Plk2-deficient tumors grew larger than control. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk2’s tumor suppressor functions in relationship to mTOR signaling. Archival specimens from 12 colorectal adenocarcinomas were stained for markers including Plk2, phosphorylated mTOR (serine 2448) and ribosomal S6 (Serine 235/236). We show that Plk2 is expressed in normal colon, with a punctate staining pattern in supranuclear cytoplasm. In colorectal adenocarcinoma, Plk2 demonstrates complete or partial loss of expression. Strong expression of phosphorylated mTOR is observed in the invasive front. Phosphorylated S6 expression partially correlates with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, in the subset of cases examined. In order to determine whether Plk2 is lost prior to the development of invasive cancer, 8 colon polyps from 6 patients were evaluated for Plk2 expression. All polyps are positive for Plk2. A Cancer Genome Atlas search identified Plk2 mutations to be infrequent in colorectal adenocarcinomas. Neither Plk2 methylation (in the gene body) nor copy number variations correlated with changes in mRNA expression levels. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR and phosphorylated S6 at the invasive front in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression. Plk2 protein expression is lost at the same stage in colorectal carcinogenesis as p53. The p53 dependence of Plk2 loss and tumor suppressor function in relationship to mTOR signaling may have therapeutic implications.

BackgroundFusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions.MethodsWe enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety.ResultsA total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events.ConclusionsLarotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

Breast cancer is a major cause of cancer-related death and there is a need for novel therapies with increased efficacy and decreased toxicity. The small molecule ONC201 was initially identified as a TRAIL pathway inducer. The compound has entered early phase clinical trials and is being tested in a range of solid tumors and hematological malignancies. Our previously published data demonstrate that ONC201 has potent anti-proliferative and pro-apoptotic effects in a broad range of breast cancer subtypes through TRAIL-dependent and TRAIL-independent mechanisms. Analogs of ONC201 with a shared pharmacophore have been developed and form the novel “imipridone” class. When compared with ONC201, imipridones ONC206, ONC212, and ONC213 showed increased potency of anti-proliferative or pro-apoptotic effects in breast cancer cells. We were interested in further defining the previously unstudied anti-proliferative effects of the imipridone compounds. Single agent efficacy of potent imipridone ONC212 in a xenograft model of TNBC was observed in the absence of apoptosis induction. This indicates that the anti-proliferative actions of the compound are sufficient for an in vivo anti-tumor effect. Our lab has previously shown that ONC201 activates an ATF4-dependent integrated stress response (ISR), essential for apoptosis induction in colon cancer cells. In contrast, in breast cancer cells, although ATF4 knockdown did not block cell death induced by ONC201 it did partially abrogate the anti-proliferative effects of the compound. The mammalian target of rapamycin complex 1 (mTORC1) is a well-known regulator of cellular growth and proliferation. mTORC1 signaling is inactivated in breast cancer cells following treatment with ONC201 and its analogs ONC212 and ONC213, regardless of whether the cells undergo apoptosis. Knockdown of ATF4 abrogated ONC201-mediated inhibition of p70 S6 kinase and ribosomal protein S6 phosphorylation, linking ISR induction to mTORC1 inhibition. We hypothesized that sestrin2, an endogenous mTORC1 inhibitor known to be upregulated following cellular stress, might represent a link between induction of ATF4 and inhibition of mTORC1. Treatment with ONC201 and its analogs ONC212 and ONC213 in multiple breast cancer cell lines resulted in sestrin2 upregulation. This was blocked by ATF4 knockdown. Furthermore, knockdown of sestrin2 abrogated the effects of ONC201 on mTORC1 signaling in breast cancers from multiple molecular subtypes. Previous mechanistic studies have focused exclusively on the relevance of ATF4 in the pro-apoptotic effects of ONC201. The novel findings described here help to elucidate the mechanism behind the potent and understudied anti-proliferative effects of the imipridones. Our findings also strengthen the preclinical rationale for testing of imipridone compounds against breast cancers regardless of molecular subtype. Citation Format: Ralff MD, Kline CLB, El-Deiry WS. Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-08.

Inflammatory cytokines, immune stimulants, chemokines, and tumor growth/growth suppressing factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact response to therapeutics. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immune synergy heat map rankings of analytes for drug and cell line combinations. We observed overall trends in drug-class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing sTRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased sTRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger data-set may be utilized in the clinic to aid in immune synergy prediction of a patient's response to therapy based on tumor genotype. Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Wafik S. El-Deiry. A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P036.

Background: 9-ING-41, a small molecule specific GSK-3 inhibitor, demonstrated favorable efficacy and safety in a Phase I clinical trial evaluating 9-ING-41 monotherapy and 8 chemotherapy combinations in 236 patients (pts) that has since been expanded to a Phase II multicenter study (NCT03678883). Interim results documented encouraging clinical activity, especially in patients with advanced pancreatic ductal adenocarcinoma (PDAC) and durable responses in melanoma and ATLL. Potential genomic biomarkers for 9-ING-41 response have not yet been evaluated. Methods: Next-generation sequencing (NGS) of tumor samples was obtained, whenever possible, for those receiving 9-ING-41 alone or in combination with chemotherapy across all sites. Genomic sequencing reports were reviewed for somatic alterations deemed potentially actionable or biologically relevant. For those with PDAC, best response was determined using RECIST 1.1 for those who completed at least two cycles of treatment. Chi-square frequency statistics were used to show the observed versus expected rate of pathogenic variants between patients with disease control (complete response [CR], partial response [PR] or stable disease [SD]) and progressive disease (PD). Results: NGS results were available for 135 pts across 11 sites. Most common histologies were PDAC (n=32), colorectal (n=17), and melanoma (n=9). Among pts with PDAC, NGS results were available for 32 pts (23 tumor samples, 9 ctDNA). Four patients received 9-ING-41 monotherapy; 28 received 9-ING-41 combined with gemcitabine/nab-paclitaxel (n=18), gemcitabine (n=7), or irinotecan (n=3). The most frequently mutated genes among PDAC included KRAS (n=22 pts), TP53 (n=21), CDKN2A (n=12), SMAD4 (n=4), CDKN2B (n=4), MTAP (n=3), ATM (n=3), AKT2 (n=2), and ARID1A (n=2). Of these 32 pts, 25 pts were evaluable for response: 2 pts had CR, 2 PR, 9 SD, and 12 PD. Among the pts with CR, one had tumor without pathogenic variants, the other displayed TP53 and KRAS mutations. Pts with PR: one tumor had no pathogenic mutations, the other tumor harbored mutations in ARID1A, TP53, FGF14, and ROS1. Of the nine patients with SD, 8 had KRAS mutations, 5 had CDKN2A loss of function, and 4 inactivating mutations in SMAD4. Eight out of 12 pts with PD had KRAS mutations. There were no significant differences between pts who had disease control and those with PD in the frequency of KRAS (χ2= 0.0189, P=0.89), TP53 (χ 2= 0.0712, P=0.78), or SMAD4 (χ 2= 1.9631, P= 0.1611). Conclusions: 9-ING-41 has shown clinical benefit in patients with PDAC independent of tumor somatic mutational profile. Preliminary analysis does not reveal pathogenic mutations that are associated with clinical benefit. Additional biomarker studies are ongoing. Citation Format: Brittany A. Borden, Andre De Souza, Devalingam Mahalingam, Steven Powell, Pamela N. Munster, Kelsey Huntington, Andrew Mazar, Ludmila Calvacante, Francis J. Giles, Wafik S. El-Deiry, Howard P. Safran, Benedito A. Carneiro. Genomic biomarkers for response to 9-ING-41, a small molecule selective glycogen synthase kinase-3 (GSK-3) inhibitor, in pancreas cancer: Preliminary results [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P021.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a 4.2% 5-year survival rate. There is an urgent need for innovative treatments for patients suffering from PDAC. Patients with PDAC often have immunosuppressed tumors that are apoptosis-resistant and have limited immune cell infiltration and/or activation. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces cancer cell apoptosis during innate immunity, while ONC212 is a potent fluorinated second-generation imipridone currently in IND-enabling studies that induces TRAIL signaling and the integrated stress response (ISR). TLY012 is a novel PEG’ylated trimeric TRAIL receptor agonist being clinically developed to overcome the short half-life of TRAIL. We hypothesized that combining a TRAIL pathway inducer and a TRAIL receptor agonist may be efficacious in PDAC and may activate immune cell killing based on prior work with imipridone ONC201. Immune cell co-culture experiments were conducted using PANC1 PDAC cells and TALL-104 CD8+ cytotoxic T-cells at a 1:1 effector-to-target cell ratio with or without ONC212. We observed a significant increase in T-cell killing of PDAC cells in the co-culture assay following treatment with ONC212 at several doses as compared to controls. We noted an increase in tumor-immune cell interactions as measured by the number of T-cells that were directly touching tumor cells at each timepoint. The BxPC-3 immunodeficient murine PDAC model revealed an increase in natural killer (NK) cell tumor infiltration by immunohistochemistry (IHC) staining for NK1.1 after 30 days of 50 mg/kg ONC212 treatment three times a week. The combination of ONC212 and TLY012 was evaluated using subcutaneously-implanted KPC-Luc PDAC cells in a syngeneic immunocompetent C57BL/6 mouse model. Mice treated with 25 mg/kg ONC212 alone twice a week, 10 mg/kg TLY012 alone twice a week, or combination therapy with ONC212 and TLY012 showed statistically significant decreases in tumor growth compared to controls after 5 weeks of treatment (n=6 mice per treatment condition) by tumor volume and in vivo bioluminescent imaging. We are analyzing murine plasma cytokine profiles, changes in tumor-infiltrating NK- and T-cells by flow cytometric and IHC analysis of murine spleen and KPC-Luc tumor samples. We report a novel immune stimulation of the TRAIL pathway and T-cell activation by ONC212 plus TLY012 leading to cytotoxic anti-PDAC responses in vivo. Citation Format: Kelsey E. Huntington, Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, Wafik S. El-Deiry. ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-064.

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201's interaction with DRD2 plays a role in ONC201's anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type–specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201's anticancer effects. Although ONC201's anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201's anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201's ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.

TP53 is the most commonly mutated gene in human cancer with over 100,000 literature citations in PubMed. This is a heavily studied pathway in cancer biology and oncology with a history that dates back to 1979 when p53 was discovered. The p53 pathway is a complex cellular stress response network with multiple diverse inputs and downstream outputs relevant to its role as a tumor suppressor pathway. While inroads have been made in understanding the biology and signaling in the p53 pathway, the p53 family, transcriptional readouts, and effects of an array of mutants, the pathway remains challenging in the realm of clinical translation. While the role of mutant p53 as a prognostic factor is recognized, the therapeutic modulation of its wild-type or mutant activities remain a work-in-progress. This review covers current knowledge about the biology, signaling mechanisms in the p53 pathway and summarizes advances in therapeutic development.

CDK4/6 targeting is a promising therapeutic strategy under development for various tumor types. In this study, we used computational methods and The Cancer Genome Atlas dataset analysis to identify novel miRNAs that target CDK4/6 and exhibit potential for therapeutic development in colorectal cancer. The 3′UTR of CDK4/6 mRNAs are targeted by a family of miRNAs, which includes miR-6883-5p, miR-149*, miR-6785-5p, and miR-4728-5p. Ectopic expression of miR-6883-5p or miR-149* downregulated CDK4 and CDK6 levels in human colorectal cancer cells. RNA-seq analysis revealed an inverse relationship between the expression of CDK4/6 and miR-149* and intronic miRNA-6883-5p encoding the clock gene PER1 in colorectal cancer patient samples. Restoring expression of miR-6883-5p and miR-149* blocked cell growth leading to G0–G1 phase cell-cycle arrest and apoptosis in colorectal cancer cells. CDK4/6 targeting by miR-6883-5p and miR-149* could only partially explain the observed antiproliferative effects. Notably, both miRNAs synergized with the frontline colorectal cancer chemotherapy drug irinotecan. Further, they resensitized mutant p53-expressing cell lines resistant to 5-fluorouracil. Taken together, our results established the foundations of a candidate miRNA-based theranostic strategy to improve colorectal cancer management. Cancer Res; 77(24); 6902–13. ©2017 AACR.

Glioblastoma is an aggressive central nervous system tumor with a 5-year-survival rate of less than 10%. Patients diagnosed with the disease are treated with surgery, radiation and temozolomide chemotherapy. Despite survival benefits, patients eventually relapse. There is a need for new treatments with improved efficacy. Imipridone ONC201 is a small molecule originally identified as a TNF-related apoptosis inducing ligand (TRAIL)-inducing compound. ONC201 has the unique ability to induce expression of both pro-death ligand TRAIL and its receptor DR5 through engagement of the cellular integrated stress response (ISR) pathway. Arrillaga-Romany et al. report early results from futility analysis of a phase II clinical trial of ONC201 in 17 patients with recurrent or refractory glioblastoma conducted at the Massachusetts General Hospital Cancer Center. The results are promising, as ONC201 shows preliminary signs of efficacy. Further testing of ONC201 in an expansion cohort of patients with glioblastoma is ongoing.

// Niklas K. Finnberg 1 , Prashanth Gokare 1 , Avital Lev 1 , Sergei I. Grivennikov 2 , Alexander W. MacFarlane IV 3 , Kerry S. Campbell 3 , Ryan M. Winters 4 , Karen Kaputa 4 , Jeffrey M. Farma 5 , Abbas El-Sayed Abbas 6 , Luigi Grasso 7 , Nicholas C. Nicolaides 7 and Wafik S. El-Deiry 1 1 Department of Hematology/Oncology and Molecular Therapeutics Program, Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Fox Chase Cancer Center, Philadelphia, PA, USA 2 Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA 4 Biosample Repository Facility, Fox Chase Cancer Center, Philadelphia, PA, USA 5 Division of General Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 6 Division of Thoracic Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 7 Morphotek Inc., Philadelphia, PA, USA Correspondence to: Wafik S. El-Deiry, email: // Keywords : 3D, organoid, colorectal cancer, immune cells, biomarkers Received : June 24, 2017 Accepted : July 20, 2017 Published : August 05, 2017 Abstract We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient’s peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo . Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G1 phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290–8. ©2017 AACR.

Hypoxia is an inherent impediment to cancer therapy. Palbociclib, a highly selective inhibitor for CDK4/6, has been tested in numerous clinical trials and has been approved by the FDA. We previously reported that CDK inhibitors can destabilize HIF1α regardless of the presence of hypoxia and can sensitize tumor cells to TRAIL through dual blockade of CDK1 and GSK-3β. To translate this knowledge into a cancer therapeutic strategy, we investigated the therapeutic effects and molecular mechanisms of CDK inhibition against colon cancer cells under normoxia and hypoxia. We found that palbociclib sensitizes colon cancer cells to hypoxia-induced apoptotic resistance via deregulation of HIF-1α accumulation. In addition to inhibition of cell proliferation, we observed that palbociclib promotes colon cancer cell death regardless of the presence of hypoxia at a comparatively high concentration via regulating ERK/GSK-3β signaling and GSK-3β expression. Furthermore, palbociclib synergized with irinotecan in a variety of colon cancer cell lines with various molecular subtypes via deregulating irinotecan-induced Rb phosphorylation and reducing HIF-1α accumulation under normoxia or hypoxia. Collectively, our findings provide a novel combination therapy strategy against hypoxic colon cancer cells that may be further translated in the clinic.

Perineural invasion (PNI) has been implicated as a poor prognostic indicator in many cancers. The National Comprehensive Cancer Network recommends consideration of observation or adjuvant therapy in the presence of PNI in early colon cancer. These recommendations are based on single institutional studies that fail to evaluate PNI within the context of adjuvant chemotherapy. The US National Cancer Database (2004-2012) was reviewed for patients with node negative colon cancer, and stratified by PNI and receipt of chemotherapy. Of 21,488 patients evaluated, 55.2% had T3 disease (n = 11,852), 23.1% had T2 (n = 4,971), 14.4% had T1 (n = 3,088), and 7.3% had T4 disease (n = 1,577); 4.6% (n = 987) had PNI. Most patients (86.8%, n = 18,641) did not have PNI and did not receive chemotherapy; 8.7% (n = 1,860) did not have PNI but received chemotherapy; 3.7% (n = 785) had PNI and did not receive chemotherapy, and 0.9% (n = 202) had PNI and received chemotherapy. Among those with PNI, patients who received chemotherapy tended to be younger (P

// Safoora Deihimi 1,2,3,7 , Avital Lev 1,2,3 , Michael Slifker 4 , Elena Shagisultanova 3,9 , Qifang Xu 2 , Kyungsuk Jung 5 , Namrata Vijayvergia 3 , Eric A. Ross 4,6 , Joanne Xiu 8 , Jeffrey Swensen 8 , Zoran Gatalica 8 , Mark Andrake 2 , Roland L. Dunbrack 2 and Wafik S. El-Deiry 1,2,3,7 1 Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Fox Chase Cancer Center, Philadelphia, PA, USA 2 Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Department of Hematology/Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 4 Biostatistics and Bioinformatics Department, Fox Chase Cancer Center, Philadelphia, PA, USA 5 Department of Medicine, Fox Chase Cancer Center, Philadelphia, PA, USA 6 Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, USA 7 Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, USA 8 Caris Life Science, Phoenix, AZ, USA 9 University of Colorado Denver Cancer Center, Denver, CO, USA Correspondence to: Wafik S. El-Deiry, email: // Keywords : BRCA2, EGFR, NTRK, colorectal cancer, MLH1 Received : April 14, 2017 Accepted : April 26, 2017 Published : May 23, 2017 Abstract Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors ( P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.

A MESSAGE FROM ASCO’S PRESIDENT I am pleased to present Clinical Cancer Advances 2017, which highlights the most promising advances in patient-oriented cancer research over the past year. The report gives us an opportunity to reflect on what an exciting time it is for cancer research and how swiftly our understanding of cancer has improved. One year ago, the White House announced the national Cancer Moonshot program to accelerate progress against cancer. This shared vision of progress has reinvigorated the research community, identified new areas of scientific collaboration, and raised our ambitions regarding what may be possible beyond the progress we have already made. When I entered the field 35 years ago, I could not have imagined where we would be today. We can now detect cancer earlier, target treatments more effectively, and manage adverse effects more effectively to enable patients to live better, more fulfilling lives. Today, two of three people with cancer live at least 5 years after diagnosis, up from roughly one of two in the 1970s. This progress has resulted from decades of incremental advances that have collectively expanded our understanding of the molecular underpinnings of cancer. There is no better current example of this than ASCO’s 2017 Advance of the Year: Immunotherapy 2.0. Over the last year, there has been a wave of new successes with immunotherapy. Research has proven this approach can be effective against a wide range of hard-to-treat advanced cancers previously considered intractable. Researchers are now working to identify biologic markers that can help increase the effectiveness of treatment and determine who is most likely to benefit from immunotherapy. This knowledge will enable oncologists to make evidence-based decisions so as many patients as possible might benefit from this new type of treatment. Each successive advance builds on the previous hard work of generations of basic, translational, and clinical cancer researchers. Importantly, the advances described in this report would not have been possible without the individuals who volunteered to participate in clinical trials as part of their treatment. To turn the promising vision of a cancer moonshot into meaningful advances, we need sustained, robust federal funding for continued research and innovation. Approximately 30% of the research highlighted in this report was funded, at least in part, through federal dollars appropriated to the National Institutes of Health or the National Cancer Institute. Without this federal investment—unique internationally in scale, duration, and impact for decades—I fear we may lose the forward momentum needed to further the progress we see highlighted in this report. Federal lawmakers can further fuel progress by advancing initiatives that facilitate the use of big data to achieve the common good of high-quality care for all patients. Such programs, like ASCO’s CancerLinQ, will rapidly increase the pace of progress and dramatically expand the reach of treatment advances to the millions of patients who are living with cancer today or who will do so in the future. This investment will yield medical, scientific, economic, and societal benefits for years to come. Much work still lies ahead. Many questions remain about how cancer develops and spreads and how best to treat it. As you read through Clinical Cancer Advances 2017, I hope you are as inspired as I am by the gains the clinical cancer research community has made over the past year and by the promise of a new era of advances just over the horizon. Daniel F Hayes, MD, FASCO, FACP ASCO President, 2016 to 2017

Muscle-invasive bladder cancer (MIBC) frequently harbors mutations in the CDKN1A gene, which encodes the tumor suppressor protein p21, with the majority of alterations truncating the peptide. The effect of these mutations is poorly understood. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle and efficiently repair DNA damage, thus proceeding down the apoptotic pathway. We used synthetic CRISPR guide RNAs to ablate the whole peptide (sg12, targeting the 12th amino acid) or the C-terminal proliferating cell nuclear antigen (PCNA)-binding domain (sg109) to mimic different p21-truncating mutations compared with a negative control (sgGFP) in bladder cancer cell lines. Loss of detectable p21 and a stable truncated p21 peptide were identified in sg12 and sg109 single-cell clones, respectively. We found that p21-deficient cells (sg12) were sensitized to cisplatin, while cells harboring distally truncated p21 (sg12 clones) demonstrated enhanced cisplatin resistance. p21-deficient sg12 clones demonstrated less repair of DNA-platinum adducts and increased γ-H2AX foci after cisplatin exposure, suggesting there was persistent DNA damage after p21 loss. p21-deficient sg12 clones were also unable to prevent the activation of CDK1 after DNA damage, and therefore, continued through the cell cycle, resulting in replication fork collapse, potentially explaining the observed cisplatin sensitization. sg109 clones were neither unable to sequester PCNA nor localize p21 to the nucleus after DNA damage, potentially explaining the chemoresistant phenotype. Our findings suggest that different CDKN1A truncations have different and perhaps disparate biology, and that there may be a duality of effect on cisplatin sensitivity depending on mutation context. Implications: Some truncating CDKN1A mutations generate a retained peptide that may have neomorphic functions and affect cisplatin sensitivity in patients with bladder cancer.

Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3β (GSK-3β), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.

CNS tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy or temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SF8628) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy or temozolomide up to 100 μM. We observed synergy between ONC201 and radiation or temozolomide by multiple assays. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. We are evaluating the relevance of mitochondrial protease ClpP, a recently described binding target of ONC201, in ONC201-induced glioma cell death. We have observed that knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated cytotoxicity. Thus, we are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers.

Heterogeneity in the p53 response to traditional chemotherapy is recognized but its significance has yet to be exploited in the clinic. Further investigation could provide insight into the relative importance of individual p53 target genes and identify unique characteristics of specific drugs, guiding therapy selection. Wild-type or p53 null HCT-116 cells were treated with oxaliplatin, cisplatin, CPT-11 (irinotecan), or 5-Fluorouracil (5-FU) at an IC50 dose for 8 hours, in triplicate. Total cellular RNA was isolated and Clariom™ D microarrays were used to measure changes in RNA expression relative to untreated cells. TAC software was used to calculate fold-changes and statistical significance. Western blot was used to confirm treatment-induced increase in p53 and to further investigate p53 target heterogeneity at the protein level. The top 100 p53-dependent genes upregulated (FC>1.5) by each drug were identified, which revealed BTG2 as the top upregulated gene after oxaliplatin, cisplatin, and 5-FU treatment. Eight genes appeared in all four lists (GPR87, FAS, TP53INP1, DCP1B, POLH, PDGFA, MDM2, and PHLDA3) but variations in magnitude of change across drugs were noted for several including GPR87, FAS, TP53INP1, and MDM2. Moreover, heterogeneity in the magnitude of change of p53 targets was observed by western blot, which also showed differential upregulation of p21 and DR5 across drug treatment conditions. The lists contained numerous genes that were unique to each treatment, with oxaliplatin regulating the highest number of unique genes (31/100). Several transcripts outside of the top 100 were also identified as unique in each drug treatment including BAX, which showed slight preferential upregulation by 5-FU. Evaluation of the p53-independent transcriptional regulation revealed a strikingly significant and unique regulation of histone modifications by 5-FU compared to the other treatments. Beyond the specific genes mentioned, variation in the magnitude of change and identification of the unique targets after each drug treatment highlights potentially important mechanistic differences between the chemotherapeutic drugs. The appearance of eight transcripts in the top 100 upregulated genes for all four drug treatments indicates their importance as well as the contribution of the p53 response to the molecular effects of chemotherapy. We are currently further evaluating the relevance of these genes and their unique contributions to the action of the chemotherapeutic drugs. Citation Format: Lindsey Carlsen, Lanlan Zhou, Liz J. Hernández Borrero, Arunasalam Navaraj, Shengliang Zhang, Christoph Schorl, Wafik S. El-Deiry. The transcriptional response in colorectal cancer cells varies across four clinically used chemotherapeutic drugs in terms of gene identity, magnitude of change, and p53 dependence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 999.

Chromosome 9p21 deletion occurs in 25% of urothelial carcinomas (UC). 9p21 includes the MTAP gene that encodes methylthioadenosine phosphorylase, a critical enzyme in the alternate pathway of adenine synthesis. MTAP-deleted UC tumors are very sensitive to pemetrexed-induced blockage of purine synthesis. MTAP is commonly co-deleted with CDKN2A, an immediately adjacent gene associated with sensitivity to CDK4/6 inhibitors. We describe clinical and molecular features of a 9p21-deleted subgroup of UC to better inform targeted therapy approaches. We retrospectively analyzed 46 patients (pts) with UC treated at our institution. All tumor specimens were submitted to next-generation sequencing (NGS). Patient demographics, clinic-pathological and genomic features, and clinical outcomes were summarized with descriptive statistics. MTAP/CDKN2A co-deletion was defined as a surrogate for 9p21 deletion. Fifteen (32%) pts with 9p21-deleted UC were identified. The median age was 68 years-old (range 54-79), 80% of pts were former or active smokers. Fifty-three percent (8 of 15) of these tumors showed squamous differentiation. Other histology variants were papillary differentiation (6), micropapillary (1), and sarcomatoid (1). Sites of tumor specimens submitted to NGS included bladder (11), lymph node (1), abdominal wall (1), renal pelvis mass (1), and bone (1). All pts had metastatic disease involving bone (9), lung (8), lymph nodes (7), liver (3), abdominal wall (2), penis (2), peritoneum (1), and pararenal mass (1). Most pts (8) underwent curative surgery and later developed metastases; seven pts presented with de novo metastases. Among 9 pts with progression-free survival (PFS) data available, median PFS was 2 months (range 1-17). Median overall survival was 6 months (range 1-35). Median PD-L1 expression on tumor cells was 1% (range 1-20); 4 tumors were PD-L1 (-). All evaluable patients (14) had stable microsatellite tumors. The most common genomic alterations co-occurring with 9p21 deletion affected CDKN2B (100%), TERT (87%), KDM6A (60%), TP53 (40%), and FGFR3 (20%). The best response associated with immune checkpoint inhibitors among 7 patients with 9p21 deletion was a disease control rate of 28.5%. Our cohort with UC and 9p21 deletion had predominantly squamous cell differentiation. Thus, MTAP is always co-deleted with CDKN2A and is a potential biomarker for pemetrexed-based combinations. Molecular characterization of 9p21 deleted UC may in the future inform targeted therapeutic approaches. Citation Format: Andre L. De Souza, Praveen Srinivasan, Luke B. Soliman, Dragan J. Golijanin, Ali Amin, Howard Safran, Anthony E. Mega, Wafik S. El-Deiry, Benedito A. Carneiro. Clinical and genomic features of advanced urothelial carcinoma with 9p21 deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1205.

Tumor cells upregulate a plethora of proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment. Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how alterations in pH modulate the interactions between immune cells and tumors cells could help elucidate opportunities for new therapeutic approaches. To determine how pH modification impacts immune cell-mediated killing of tumor cells, co-culture experiments were conducted with GFP+ SW480 colorectal cancer cells and either NK-92 natural killer cells or TALL-104 T cells at various pHs in a 48-well plate. Manipulation of pH occurred using the compounds lactic acid and sodium bicarbonate. We observed that both NK-92 cells and TALL-104 cells exhibited decreasing amounts of cell killing in response to decreasing pH. Next, to determine if therapeutic manipulation of pH via alteration of pH pump activity could overcome the negative changes in immune cell activity associated with pH, we added Cariporide, a selective NHE1 inhibitor, to the co-culture experiments. Treatment with Cariporide bolstered T cell killing in a dose-dependent manner, however the doses used did not impact natural killer cell-mediated killing. Given the importance of cell signaling on immune function, and because we saw potentially significant changes in immune killing with Cariporide, we then evaluated how changes in pH using Cariporide affected these signaling molecules. Colorectal cancer cells (HCT116, HT29, KM12C) were treated with Cariporide at 5 µm and 10 µm doses and cancer cell culture supernatants were collected and frozen down at -80 °C. Cytokine, chemokine, and growth factor levels were analyzed using a Luminex 200 multiplexing instrument. Overall cytokine levels showed a decreasing trend in response to increasing doses of Cariporide. There were notable decreases in the levels of IL-8/CXCL8, VEGF, CCL3/MIP-1 alpha, and M-CSF with increasing doses of Cariporide. All of these factors, as well as extracellular acidosis, are known to promote epithelial mesenchymal transition (EMT), thus, promoting metastatic dissemination. A therapeutic that targets factors that lead to EMT could help prevent metastasis. Moreover, we saw that pH impacts the function of immune cells and this is relevant in the context of a highly acidic TME. Therapeutically modifying the pH of the TME using Cariporide treatment may be a way to improve immune response and prevent EMT. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response. Citation Format: Kelsey E. Huntington, Aaron W. Maxwell, Anna Louie, Shengliang Zhang, Lanlan Zhou, Wafik S. El-Deiry. Decreasing colorectal cancer extracellular pH dampens immune cell killing and is partially ameliorated by treatment with NHE1 inhibitor Cariporide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2736.

ONC201, a first-in-class, small molecule, imipridone therapy, is known to induce apoptosis via upregulation of TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic death receptor (DR5), independent of p53. Current trials have been exploring ONC201 as a monotherapy or in conjunction with radiotherapy, in treatment of patients with newly diagnosed and refractory diffuse intrinsic pontine gliomas (DIPG), a disease which remains incurable at present. We sought to identify synergy between ONC201 or second generation impridones (ONC206 and ONC212), and other chemotherapeutics with known promising pre-clinical activity against DMGs. Seven patient derived H3K27M mutant DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to ONC201, ONC206, or ONC212 either as monotherapy or in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Dose dependent response to imipridone therapy was noted in all cell lines, with mean half maximal inhibitory concentration (IC50) of 1.46 µM in ONC201, 0.11 µM in ONC206, and 0.03 µM in ONC212. Synergy was identified via combination studies, with combination indices of Citation Format: Robyn Borsuk, Lanlan Zhou, Yiqun Zhang, Varun V. Prabhu, Joshua E. Allen, Nikos Tapinos, Rishi Lulla, Wafik S. El-Deiry. Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 635.

Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1044.