Single-agent Treatment Research Articles

Abstract 7223: A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma

Abstract KRAS mutations drive over 90% of pancreatic cancers, but KRAS has been proven difficult to target. Downstream of KRAS, MEK inhibition in combination with BCL-XL inhibition is synthetically lethal and an effective treatment for gynecologic, but not colorectal malignancies. Like colorectal cancer, pancreatic cancer often exhibits aberrant HER/EGFR activity. We hypothesized that a triplicate regimen targeting MEK, BCL-XL, and EGFR/HER would be effective for pancreatic cancer. Trametinib (MEK inhibitor), Afatinib (pan-EGFR/HER inhibitor), and Navitoclax (BCL-XL inhibitor); or DT2216 (a BCL-XL/BCL-1 proteolysis targeting chimera, PROTAC) were explored using various combinatorial strategies. These drug combinations were tested using four primary human pancreatic cancer cell lines using live cell imaging (Incucyte) and an in vivo murine model. Group comparisons were analyzed using one-way ANOVAs, and adjusted p-values <0.05 were considered significant. We observed that no single-agent treatments were effective. MEK inhibition plus anti-BCL-XL (Trametinib + Navitoclax or Trametinib + DT2216) demonstrated efficacy in one of the four cell lines. We noted synergistic effects on reduced cell growth and increased cell death when EGFR/HER inhibition was included by the administration of Afatinib. The triplet combination resulted in synergistic effects in three of four cell lines with Navitoclax and four of four cell lines with DT2216 (q<0.05). This effect was confirmed with DT2216 inhibition of BCL-XL in an in-vivo murine model. In conclusion, our study shows that a combinatory strategy targeting MEK, BCL-XL, and pan-EGFR/HER receptor kinase shows significant synergistic effects in pancreatic cancer. Clinical trials that assess the efficacy and safety of this strategy for patients with KRAS-mutant pancreatic cancer are warranted. Our data also suggests that novel KRAS inhibitors as monotherapy are likely to fail. Citation Format: Gerik W. Tushoski-Alemán, Alexandra J. Crespin, Chibeze J. Oguejiofor, Jordan A. McKean, Kelly M. Herremans, Thomas J. George, Carmen J. Allegra, Steven J. Hughes, Song Han. A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7223.

Abstract 5379: Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC

Abstract BACKGROUND: Inactivating mutations and deletions in STK11 are present in 9-20% of NSCLC and result in immunologically cold tumors that respond poorly to therapy, despite having a high tumor mutational burden and neoantigen load. STK11 mutations in NSCLC are associated with higher levels of ULK1/2-mediated macroautophagy, resulting in localization of MHC-I to autophagosomes and lysosomes leading to its degradation and decreased antigen presentation. Knockdown of ULK1/2 can restore surface MHC-I in highly autophagic cell lines. Here, we show that inhibition of ULK1/2 decreases macroautophagy, resulting in increased antigen processing and presentation (APP) and MHC-I levels in cancer cells, supporting the hypothesis that inhibition of ULK1/2 will restore antigen presentation in NSCLC STK11mut tumors. METHODS: TCGA datasets were analyzed to determine transcriptional differences in autophagy and APP between STK11 WT and mutant NSCLC tumors. CRISPR was used to stably knockout STK11 in multiple human NSCLC cell lines to generate isogenic pairs. mRNA and protein levels of APP machinery in NSCLC cell lines were assessed using qPCR, western blots, and flow cytometry. CRISPR was additionally used to knockout ULK1 and ULK2 in human STK11 mutant A549 cells. ULK1/2 tool compounds were synthesized based on existing patent literature. Effects of the combination of ULK1/2 inhibition with low levels of IFNγ on surface MHC-I levels were evaluated in several NSCLC cell lines by flow cytometry. RESULTS: NSCLC tumors with loss of function mutations or deletion of STK11 display increased transcription of genes in the ULK complex and reduced transcription of genes necessary for APP. STK11mut and KO NSCLC cell lines also display lower levels of APP at the mRNA and protein levels. Double knockout of ULK1 and ULK2 in A549 cells is not cytotoxic and results in decreased autophagy as well as increased immunoproteasome components and cell surface MHC-I. ULK inhibition reduces autophagy and restores APP at the protein level, but not at the transcriptional level, in all NSCLC cell lines evaluated. Combination of very low concentrations of IFNγ with ULK inhibition demonstrates additive increases in MHC-I greater than either single-agent treatment alone. CONCLUSIONS: STK11mut/del is associated with poor prognosis in NSCLC and leads to increased ULK-induced autophagy and decreased APP. ULK inhibition increases APP and surface MHC-I levels in STK11mut cell lines. In the presence of low concentrations of IFNγ, ULK1/2 inhibition restores APP in NSCLC STK11mut cell lines to the levels seen in NSCLC STK11 WT cell lines. The data presented here provides a rationale for targeting ULK1/2 to amplify immune recognition in immunologically cold tumors with high autophagy to increase responses to immunotherapies. Citation Format: Samantha C. Schwager, Eugene Park, Anne Van Abbema, Yihong Guan, Connor Rosen, Sean Cho, Michael Black, Inna Kurilyak, Jenna Jeffrey, Juan Jose Fung, Susan L. Paprcka, Ester Fernandez-Salas. Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5379.

Abstract 659: TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro

Abstract Introduction: Ferroptosis is an emerging potential target for the treatment of colorectal cancer (CRC) and has been shown to sensitize cancer cells to combinatory chemotherapy. Our studies show that the TR-107 compound, an agonist of ClpP - the proteolytic subunit of mitochondrial maintenance complex - can synergize with Erastin, an inducer of ferroptosis cell death. TR-107 potently disrupts mitochondrial proteostasis and attenuates oxidative phosphorylation, resulting in excessive oxidative stress. Here, we show that Erastin synergizes with TR-107 activity by downregulating the cofactor of GPX4, a major intracellular antioxidant defense resulting in CRC cell apoptosis. Methods: We first evaluated the efficacy of TR-107 against eight CRC cell lines belonging to four established consensus molecular subtypes. Cells were treated with control (DMSO) or TR-107 (10 nM, 50 nM, 1 µM) for 24, 48, and 72 hours. We determined IC50 values with Cell Titer-Glo, cell proliferation with the Beckman Coulter cell counter, cell cycle with flow cytometry, OXPHOS activity with Seahorse Real-Time ATP Rate assay, mitochondrial protein levels with western blot and proteomics, and differential gene expression using RNA sequencing and RT-PCR. Subsequently, we assessed the efficacy of TR-107 and Erastin co-treatment. Cells were treated with DMSO, TR-107 (10, 50 nM), Erastin (1, 10 µM), or combinations of the abovementioned concentrations. Cell viability was measured 72 hours post-treatment with Cell Titer-Glo. Synergy scores (HSA) were calculated with Synergyfinder on RStudio. Ferroptosis-related protein expression 24 hours after co-treatment was assessed with western blot and densitometry. Results: TR-107 IC50 values ranged from 2-70 nM, producing a dose- and time-dependent inhibitory effect in cell proliferation. Western blot showed that 50 nM TR-107 abolished the expression of critical mitochondrial proteins ClpX, TFAM, and TUFM. Seahorse data showed a significant reduction in OXPHOS activity. Multi-omics revealed downregulation of respiratory chain complex subunits, mtDNA transcription/translation factors, and upregulation of mitophagy and ferroptosis-related pathways in treated cells. Co-treatment with Erastin produced synergistic HSA scores in 63% (5/8) of assessed CRC cell lines. RKO and NCI-H508 were highly sensitive to Erastin treatment, suggesting that lower Erastin concentrations may synergize with TR-107 and thus reduce dose toxicity. Western blot analysis 24 hours post-treatment showed a loss of GPX4 protein levels in co-treated cells compared to 50 nM TR-107 treatment. This suggests a rapid downregulation of intracellular antioxidant defense that may induce ferroptosis sooner and more effectively than single-agent treatment. Conclusion: In vitro results suggest that co-treating with TR-107 and Erastin produces a significant, synergistic reduction in CRC cell viability. Citation Format: Michael Giarrizzo, Joseph F. LaComb, Hetvi R. Patel, Agnieszka B. Bialkowska, Rohan Reddy, Lee M. Graves, Edwin J. Iwanowicz. TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 659.

Abstract 6491: Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung cancers

Abstract KRAS and LKB1 mutations (KL) in non-small cell lung cancer (NSCLC) patients often results in poor prognosis, a high tumor mutational burden, and resistance to conventional therapy. The metabolic changes with these mutations can create unique vulnerabilities to be targeted therapeutically. HDAC inhibition has been reported as a promising approach to overcome MEK inhibitor resistance in animal models of melanoma, pancreatic cancer, and KL mutant NSCLC. Inhibition of HDAC3 and HDAC6 have independently been shown to have therapeutic activity in KL NSCLC. We now report on the activity of two novel HDAC3/6 inhibitors in cell culture and mouse models of human KL (A549) compared to KRAS/TP53-mutant NSCLC (KP) (CALU-6) alone and individually combined with the MEK inhibitor trametinib. GB-1101 inhibits HDAC3 and HDAC6 and GB-3103 inhibits HDAC1,3,6,10. SynergyFinder multi-dose combination data analytics showed synergistic interactions for both GB-1101 or GB-3103 when combined with trametinib in KL mutant A549 cells with maximum synergy scores of δ = 9.24 and 4.21, using zero interaction potency (ZIP) for synergy scoring. GB-1101 or GB-3103 when combined with trametinib in KP mutant CALU-6 cells did not demonstrate synergistic interaction with synergy scores of δ = -1.58 and -0.53, respectively. Mice were inoculated subcutaneously with 5.0x106 A549 or CALU-6 cells and treatment with GB-1101 (75 mg/kg qd, oral), GB-3103 (5.0 mg/kg qd, subcutaneous), trametinib (1.0 mg/kg qd, oral) and each drug in combination with trametinib was initiated when tumors reached an average of 120mm3. Single agent and combination treatments were well tolerated. GB-1101 exhibited tumor growth inhibition (TGI) of 33.0% in the A549 (KL) compared to 8.5% in CALU-6 (KP). In combination with trametinib, GB-1101 showed increased activity (TGI=92.7%) compared to trametinib alone (TGI=69.5%) in KL mutant A549. In KP mutant CALU-6 tumors, combination benefit was not observed; combination of GB-1101 and trametinib yielded a TGI=93% equivalent to trametinib alone (TGI=91%). GB-3103 resulted in a TGI of 66.7% in the KL mutant A549 and TGI= 55.9% in KP mutant CALU-6. In KL mutant A549, GB-3103 in combination with trametinib, showed increased inhibition (TGI=94.3%) compared to trametinib alone (TGI=69.5%). No combination benefit was seen for GB-3103 in KP mutant CALU-6 with the combination treatment TGI of 93.1% compared to trametinib alone TGI=90.7%. These results confirm and extend the finding that for KL mutant NSCLC, HDAC3/6 inhibitors show therapeutic benefit in combination with trametinib that is not observed in KP tumors. These data reveal a promising epigenetic-based combinatorial strategy for treating lung cancers harboring concurrent KRAS/LKB1 mutations, a path for clinical translation, and the potential to improve treatments for KRAS/LKB1 mutant NSCLC patients where there are currently no robust therapeutic strategies. Citation Format: Matthew Zucker, Kendra Devore, Logan Chesney, Gregg Hirschfeld, Annissa Reyna, Betsy Linnane, Joseph Mascarenhas, Maggie Murray, Alan Miller, Paul Gonzales, Stephen Gately. Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6491.

Abstract 7196: Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines

Abstract Background: Endocrine therapy (ET) is the standard of care for hormone receptor positive (HR+) breast cancer (BC), but ET resistance develops in most patients (pts). One resistance mechanism involves cross talk between the PI3K-AKT-mTOR and estrogen receptor (ER) signaling pathways, resulting in increased ER signaling and tumor proliferation. This is mitigated by mTOR inhibition as demonstrated by improved outcomes with exemestane plus the mTOR inhibitor (mTORi) everolimus in pts with HR+ advanced BC. While PI3K inhibitors (PI3Ki [e.g. alpelisib]) are efficacious in pts with HR+, PIK3CA-mutated BC, resistance can occur due to acquired genomic alterations in the PI3K-AKT-mTOR pathway. Combination therapy that targets a critical downstream node, mTOR, could delay or overcome acquired resistance due to activation of upstream components of the PI3K-AKT-mTOR pathway. nab-Sirolimus is an intravenously administered, albumin-bound nanoparticle form of the mTORi sirolimus, with significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects than oral mTORis in nonclinical tumor models. We evaluated the antiproliferative and cytotoxic effects of nab-sirolimus in combination with ET or PI3Kis in BC cell lines. Methods: HR+, PI3K-mutated BC cell lines (MCF7 or MDA-MB-361) were incubated for 5 days with increasing concentrations of nab-sirolimus and the selective ER degrader fulvestrant. MDA-MB-361 cells or HR-negative (HR−), PI3K-mutated BC cells, MDA-MD-453, were incubated for 5 days with increasing concentrations of nab-sirolimus and PI3K-AKT-mTOR pathway inhibitors gedatolisib or alpelisib. Antiproliferative/cytotoxic effects of single-agent and combination treatment were assessed via an automated trypan blue exclusion assay. Results: Addition of 20 or 80 nM nab-sirolimus to fulvestrant (12.5-500 nM) markedly decreased cell viability and nearly doubled cell death in MCF7 and MDA-MB-361 cells. Antiproliferative effects of low-dose gedatolisib (2.5-10 nM) were enhanced by 61-71% when nab-sirolimus (20 or 80 nM) was added to MDA-MB-453 cells. While low doses (2.5, 5, and 10 nM) of gedatolisib alone led to limited cytotoxicity (4.2-9.6%), up to 48.9% of cell death occurred when 20 or 80 nM nab-sirolimus was added. Results were similar with alpelisib (1 uM) plus nab-sirolimus (20 or 80 nM). Western blot showed increased p4EBP1 in response to fulvestrant or PI3Ki treatment, which was reversed with the addition of nab-sirolimus. Conclusions: nab-Sirolimus enhanced the cytotoxic effects of fulvestrant in HR+ BC cells and of PI3Kis in HR+ and HR− BC cells. Addition of nab-sirolimus to ET or other PI3K-AKT-mTOR pathway inhibitors may overcome mechanisms of resistance; further exploration is warranted to elucidate clinical relevance. Citation Format: Khine Nyein Myint, Sean Wallace, Shihe Hou, Maria Zalath, Brian McMorran, Andrew Kwon, Igor Vivanco. Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7196.

Abstract 2609: Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors

Abstract We have recently shown that targeting circulating LGALS3BP with a maytansinoid-based non-internalizing antibody-drug conjugate (ADC) resulted in tumor eradication of high-risk NMYC-amplified neuroblastoma patient-derived xenografts. LGALS3BP is a high molecular weight, hyperglycosylated and secreted protein which is enriched at the surface of cancer-derived extracellular vesicles (EVs). Here, using a neuroblastoma pseudometastatic preclinical model, we extended our previous work demonstrating that EVs-associated LGALS3BP strictly correlates with disease progression and dissemination. Moreover, to investigate whether LGALS3BP ADC, named 1959sss/DM4, possesses immune-modulating properties, we analyzed DM4 ability to induce immunogenic cell death (ICD). Interestingly, using NXS2 murine neuroblastoma cells, we found that DM4 induced a time-dependent surface expression of three different ICD marker, i.e. calreticulin, HSP70 and HSP90. As it was recently demonstrated that to limit immune surveillance NXS2 cells increase PD-L1 expression in response to IFNγ production in the tumor immune infiltrate, we evaluated potential synergism between LGALS3BP targeting ADC and an anti-PD1 immune check-point inhibitor. To this aim, we genetically manipulated NXS2 cells which resulted nearly negative for murine LGALS3BP expression. Therefore, using a lentivirus-based gene transduction system, we obtained NXS2 cells producing large amounts of vesicular human LGALS3BP. Of note, the vesicular LGALS3BP could be quantified by ELISA and imaged by confocal microscopy using 1959 antibody. Strikingly, while single agent treatment showed a limited antitumor activity in NXS2-LGALS3BP syngeneic model, the LGALS3BP ADC/anti-PD1 combination resulted in a marked inhibition of tumor growth, which was followed by a prolonged survival. In conclusion, our findings establish that vesicular LGALS3BP is a potential biomarker for liquid biopsy and reveals this protein as a suitable target for therapeutic strategy that combines 1959sss/DM4 ADC with immune checkpoint blockade for the treatment of LGALS3BP expressing neuroblastoma. Citation Format: Gianluca Sala, Emily Capone, Ilaria Cela, Giulio Lovato, Alessia Lamolinara, Manuela Iezzi, Cosmo Rossi, Vincenzo De Laurenzi, Rodolfo Ippoliti, Stefano Iacobelli. Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2609.

Abstract 6564: SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. First-line treatment of AML patients consists primarily of chemotherapy, sometimes combined with a targeted therapy. However, elderly patients unfit for chemotherapy receive venetoclax, a BCL2 inhibitor, combined with azacitadine or decitabine. While responses in these patients are high (50-70%, with median OS of 10-17 months), around 30-50% of them do not respond and most relapse within 1-10 years. Increased MAPK pathway activity or low frequency RAS pathway mutations have been identified as key mediators of resistance to standard-of-care targeted therapies for AML, including venetoclax. Here, we found that in DepMap whole-genome CRISPR screens, leukemia cell lines, including AML, show a particular dependency on SOS1. A subsequent PRISM screen with a selective inhibitor of SOS1 confirmed these results. To validate these results in vitro, we treated 7 AML cell lines showing strongest dependency on SOS1 based on DepMap and PRISM screens, with SOS1i or a SHP2 inhibitor. We found that in 4 of 7 cell lines treatment with SOS1i or SHP2i strongly impaired proliferation. In 3 of 4 responding cell lines, the anti-proliferative effect with either SOS1i or SHP2i was stronger than azacytidine single agent treatment. We next treated 29 primary ex vivo cultures of AML with SOS1i and found that the treatment induced strong or intermediate anti-proliferative effect in 21 of 29 models (72%). In two AML PDX models, treatment with SOS1i as a single agent resulted in approximately 25% reduction of human CD45+ AML blast cells in both models. Venetoclax treatment led to approximately 75% decrease in AML blasts, while the combined venetoclax and SOS1i treatment resulted in near complete eradication of the disease. Annexin V staining of AML monoblasts demonstrated that this decrease was due to an increase in apoptosis in the combination groups. In summary, we demonstrate that SOS1 inhibitor treatment may be used to improve responses to venetoclax in AML patients. Citation Format: Kaja Kostyrko, Melanie Hinkel, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Daniel Gerlach, Irene Waizenegger. SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6564.

Abstract 5305: M9657, a tumor-targeted anti-CD137 agonist, induced significant antitumor immunity in combination with anti-PD-1 antibody

Abstract Background: M9657, a first-in-class tumor-targeted conditional immune agonist, was developed to boost antitumor immune responses in the tumor microenvironment (TME) by targeting CD137. The immune agonism potency of M9657 was validated through in vitro assays and in vivo efficacy studies in syngeneic tumor models using the surrogate antibody FS122m. Combining CD137 agonists and antiPD(L)-1 antibodies presents a novel therapeutic strategy to overcome immune checkpoint inhibitor (ICI) resistance and enhance antitumor activity via complementary mechanisms. Anti-PD-(L)-1 antibodies can reverse the suppressive immune signals, and CD137 agonists can boost T cell activation, improving antigen presentation and cytokine release. Methods: The antitumor immunity of M9657 + pembrolizumab (anti-PD-1 antibody) combination therapy was assessed using in-vitro luciferase and ex-vivo functional assays. In vivo antitumor efficacy was investigated with the M9657 surrogate (FS122m) and anti-mPD-1 in syngeneic tumor models expressing Mesothelin (MSLN). Results: The combination of M9657 and pembrolizumab displayed dose-dependent immune agonism potency and significantly enhanced tumor cell cytotoxicity and CD8+ T cell cytokine release relative to single agent treatments. In various syngeneic tumor models, the combination of FS122m with antimPD1 significantly increased antitumor efficacy relative to monotherapies, with more mice achieving complete tumor regression and prolonged median survival. Tumor-cured mice after combination treatment exhibited resistance to the primary tumor rechallenge, indicating the generation of tumor antigen-specific antitumor immunity and long-term immune protectivity. Conclusion: The combination of M9657 with anti-PD-(L)1 could overcome primary ICI therapy resistance and improve the antitumor efficacy of current anti-PD-(L)1 therapy in the clinic. Our findings support a combination strategy to combine M9657 with anti-PD-(L)1 in a clinical setting, potentially leading to enhanced antitumor immunity and improved patient outcomes. Future studies should investigate the safety and efficacy of this combination therapy in human patients. Sponsored by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945) Citation Format: Chunxiao Xu, Sireesha Yalavarthi, Hong Zhang, Uma Mahesh Gundra, Clotilde Bourin, Laura Helming. M9657, a tumor-targeted anti-CD137 agonist, induced significant antitumor immunity in combination with anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5305.

Abstract 585: Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model

Abstract Purpose: In the United States, colorectal cancer (CRC) is the second leading cause of cancer mortality. Metastatic CRC (mCRC) refractory to traditional chemotherapy is managed with regorafenib, a multiple-kinase inhibitor. However, patients show only a modest improvement in overall survival but experience high drug toxicity, adverse side effects, and poor tolerability. Thus, to reduce regorafenib-induced toxicity and to enhance antitumor activity, we combined it with a hybrid/dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition in a single agent. With syngeneic mice, the efficacy and impact on immunomodulation of this drug combination was assessed. Methods: To assess effects of the agents, C57BL/6 immunocompetent mice were injected with MC38 murine CRC cells and treated with regorafenib (6mg/kg body wt) or JAK/HDACi (30mg/kg body wt), and their combination every third day for 21 days. Upon completion of the experiment, the tumors were harvested and processed for RNA and protein expression profiles. The effect of drug treatment on immune response was analyzed by nCounter Gene Expression assays (NanoString Technologies). Cytokines were measured in serum using the MesoScale Discovery mouse V-Plex Proinflammatory Panel kit. For pharmacokinetic studies, plasma samples were assayed from C57BL/6 treated with drugs after 1, 7, 21, and 48 hrs of treatment. Results: The combination treatment significantly reduced tumor growth (volume and weight),relative to the vehicle control or single treatments. Gene expression showed higher CD45 abundance score in the combination treatment. This is an exciting observation, as CD45 has phosphatase activity and dephosphorylates key targets of the drugs used in the study. Additionally, after treatment with the combination, there was higher expression of Gzmb and Gzme and lower expression of Cx3cr1, indicating enhanced immuno modulation relative to regorafenib alone. We confirmed these findings by immunoprofiling of tumors. Higher CD45 staining, noted in tumors of mice treated with the combination, corroborated the gene expression results. Pronounced CD8 T lymphocytes infiltration was observed in combination, compared to regorafenib alone. Reduction of proinflammatory cytokine, TNF-α was noted in the combination group. This is an interesting observation as CD45 negatively regulates TNF, and combination treated mice had highest number of CD45 positive cells. Pharmacokinetic studies showed that the bioavailability of regorafenib was elevated after combination treatment relative to single-agent treatment. Conclusions: Relative to the single agents, the combination of regorafenib with JAK/HDACi was more effective, with an elevated antitumor immune response and with sustained inhibition of tumor growth. A clinical trial to evaluate this combination for the treatment of mCRCs is warranted. Citation Format: Prachi Bajpai, Farrukh Afaq, Sameer Al Diffalha, Sumit Agarwal, Hyung Gyoon Kim, Dennis Otali, Sooryanarayana Varambally, Ashish Manne, Ravi Paluri, Moh’d Khushman, Upender Manne. Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 585.

Abstract 5079: Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor models

Abstract The development of immune checkpoint inhibitors (ICIs) and combinations of immuno-oncology therapies with standard-of-care (SoC) chemotherapeutic agents has shifted therapeutic paradigms for patients with multiple cancer indications. For many patients, these combinations have shown a beneficial effect on clinical outcomes and disease management. However, additional combination therapies are still needed. Preclinically, a comparison of responses from SoC chemotherapies and ICI combinations in multiple disease models may be helpful in identifying optimal treatment combinations in the clinic. Here we report the results of multiple SoC/ICI in vivo combination efficacy studies in a large panel of mouse syngeneic models spanning seven tissue origins: 1) Colon: CT26, MC38; 2) Breast: EMT6, 4T1; 3) Kidney: Renca; 4) Bladder: MB49, MBT2; 5) Skin: B16F10, CM3; 6) Lung: TC1, LL/2, M109; 7) Brain: GL261. The advantage of using syngeneic models is not limited to the rapid and reproducible tumor growth, but importantly that the animals also have an intact immune system and clinically relevant tumor microenvironment, which are required for ICI combination studies. We tested > 10 chemo SoC combinations with a murinized anti-PD-1 antibody (DX400) in 14 syngeneic mouse models. We found that multiple SoC regimens combined well with DX400 treatment, including FOLFIRI, FOLFOX (colon), Carboplatin/Paclitaxel, Carboplatin/Pemetrexed (lung), Capecitabine/Docetaxel, Cisplatin/Gemcitabine (bladder), Cisplatin/5-FU/or capecitabine, and Oxaliplatin/5-FU/or capecitabine (gastric). Specifically, we observed that the SOC/ICI combinations resulted in enhanced antitumor activities compared to single-agent treatments in many of the models. These data, particularly specific SoC combination regimens that combine well with ICIs in each disease, are useful for optimizing promising combination approaches for clinical development and will be presented here. Citation Format: Eunsil Park, Marlene Hinton, Mingmei Cai, Malgorzata A. Gil, Gustave Hebert, Long Cui, Doug Linn, Brian J. Long, Evan R. Barry. Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5079.

Abstract 2037: Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment

Abstract Background: Human high-grade gliomas are aggressive brain cancers known for their resistance to treatment. Targeted therapies with single agents have shown limited success, and the presence of tumor heterogeneity complicates treatment. Combining multiple chemotherapeutic agents has emerged as a potential strategy to overcome these challenges. 2-deoxy-D-glucose (2-DG) is a glucose analog known for its ability to disrupt glycolysis, protein folding, and the pentose phosphate pathway. This disruption renders cancer cells more susceptible to further damage from reactive oxygen species and DNA damage. Previous studies have demonstrated synergistic effects when combining 2-DG with chemotherapeutic drugs in various cancers. Our study examines combining 2-DG with DNA-damaging agents for high-grade gliomas to uncover potential synergies, theorizing that this combination enhances cell death and metabolic stress, offering insights for better treatments against these tumors. Methods: The cell proliferation, clonogenic growth, and cell migration assays assessed viability, clonogenic formation, and migration. Additionally, the cell cycle and reactive oxygen species analysis were employed to evaluate cell division, oxidative stress, and antioxidant capacity. The Illumina NovaSeq 6000 platform measured transcript levels, and Liquid Chromatography-High-Resolution Mass Spectrometry analyzed metabolite changes in high-grade glioma cell lines following 2-DG treatment. The Agilent Seahorse Mito Stress Test evaluated alterations in cellular bioenergetics. SYNERGYFINDER 3 quantified synergistic effects of 2-DG with DNA-damaging agents, while the Annexin V-FITC Conjugates and a ROS multiplex assay assessed cell death and metabolic stress. Results: Our findings demonstrate that when used as a single agent, 2-DG induces cell senescence and limits cell migration but is insufficient to trigger cell death. Moreover, 2-DG treatment shifts glioma cells towards oxidative phosphorylation for energy production, elevates ROS levels, and hampers DNA synthesis and repair mechanisms. Our synergy study reveals promising synergistic effects when combining 2-DG with DNA-damaging agents, including 5-FU, Methotrexate, Gemcitabine, and Lomustine. This combined therapy leads to significant ROS generation and increased cell death compared to single-agent treatments. Furthermore, prolonged exposure to 2-DG leads to increased levels of both glucogenic and ketogenic amino acids within glioma cells, potentially serving as an alternative energy source. This finding hints at a prospective therapeutic approach for targeting drug-resistant cells. Conclusions: Our results suggest that the anticancer therapeutic effect of 2-DG is enhanced when used in combination with DNA-damaging agents, indicating this strategy as a potential approach for treating human high-grade glioma. Citation Format: Tracy Miller, Esther Jane, Matthew Halbert, Taylor Gatesman, Stacey Wendell, Dinesh Mohanakrishnan, Sameer Agnihotri, Daniel Premkumar, Ian Pollack. Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2037.

Abstract 2114: Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor

Abstract Background: EGFR tyrosine kinase inhibitors (TKIs) have significantly improved the survival rate and the quality of life of the NSCLC patients with EGFR mutation in the first line setting. For example, Osimertinib, a potent third-generation EGFR TKI, has demonstrated exceptional efficacy in metastatic NSCLC with specific EGFR mutations. Despite its success, the emergence of drug resistance, often linked to elevated HER3 protein expression, remains a significant challenge. U3-1402 (HER3-Dxd), an HER3 ADC, demonstrates a promising result in managing Osimertinib-resistant NSCLC. It has been reported that combining U3-1402 with Osimertinib has shown enhanced efficacy in resistant cell lines and mouse models. Here, we explore a novel HER3 dual-payload ADC (HER3 dpADC), uniquely combining a Topoisomerase I inhibitor and an EGFR TKI within a single antibody structure using the enzymatic site-specific conjugation platform developed by GeneQuantum (GQ). This innovative approach demonstrates potent and synergistic anti-tumor effects in vitro and in vivo, presenting an alternative therapeutic strategy for EGFR-mutant NSCLC patients. Results: GQ's dpADC platform technology employs two orthogonal enzymatic site-specific conjugations and stable linker technologies to efficiently generate the dual-payload ADC with high homogeneity and quality. For HER3 dpADC, HER3 Ab was conjugated with a novel Topoisomerase I inhibitor, TopoIx, and an EGFR tyrosine kinase inhibitor. In vitro DAR analysis revealed high linker stability, with minimal free payload release even after 96 hours of plasma incubation. Binding affinity assessments confirmed comparable affinity between HER3 dpADC and HER3 mAb in specific EGFR exon 19 deletion cell lines. Using various NSCLC cell lines in a 3D-spheroid culture system, HER3 dpADC demonstrated significant dose-dependent and synergistic anti-tumor activities. A robust bystander killing efficacy was observed in both HER3+/HER3- HEK293T cell coculture assays. Evaluation in CDX mouse models consistently demonstrated heightened in vivo efficacy by HER3 dpADC, aligning with the promising in vitro data. In NSCLC patient-derived xenograft (PDX) mouse models representing EGFR tyrosine kinase inhibitor-sensitive or resistant phenotypes, HER3 dpADC induced tumor regressions with no obvious toxicity, underscoring its potential as the next-generation HER3 targeting agent. Conclusion: The innovative HER3 dpADC, leveraging an efficient dual enzymatic site-specific conjugation, exhibited robust anti-tumor efficacy in both in vitro and in vivo settings, surpassing single-agent treatments. Its synergistic mechanism of action presents a promising possibility for developing a more potent and the front-line therapeutic solutions in NSCLC patients who have progressed on standard therapies. Citation Format: Lina Wang, Meijun Xiong, Xinju Gao, Chengang Zhou, Yu Han, Yanchun Li, Junhao Wang, Lili Shi, Gang Qin, Paul H. Song. Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2114.

Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. More than 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response and subsequent tumor recurrence is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration, and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration, and chemotherapy response, and whether targeting of AXL with small-molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDX), primary cultures and cell line models of RMS, consistent with its stem cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacologic blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong antitumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor burden compared with single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in patients with RMS.

Abstract B009: Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer

Abstract Ovarian cancer has a high propensity to develop therapy resistance. Epigenetic therapies can sensitize ovarian cancer cells to first-line treatments such as platinum-based chemotherapy and Poly (ADP-ribose) polymerase inhibitors (PARPi). Particularly, the reduction of histone 3 lysine 9 dimethylation (H3K9me2) via a euchromatin histone methyltransferase inhibitor (EHMTi) has been shown to sensitize PARPi-resistant cells to PARPi therapy. Additionally, cell growth defects were observed in PARPi-resistant cells treated with dual PARPi/EHMTi treatment compared to DMSO or single agent treatments. To determine the mechanism by which dual therapy sensitizes cells and reduces cell viability, bulk RNA-sequencing was performed on several PARPi-resistant cell lines treated with DMSO, PARPi, EHMTi, or dual PARPi/EHMTi. Transcriptomic analyses revealed that dual therapy increased interferon (IFN) signaling-related genes compared to DMSO or single agent treatments. Additionally, expression of transposable element (TE) transcripts, which can form double-stranded RNA (dsRNA) and induce an IFN response, was also increased in dual treated cells compared to controls. To determine if the reduction in cell viability is dependent on TEs and IFN signaling (i.e., viral mimicry), CRISPRi technology was used to silence intracellular sensors of dsRNA, RIGI and MDA5. Cell growth assays of these silenced cells showed that the reduction in cell growth observed with dual treatment was partially dependent on MDA5 expression. Based on the observed increase in IFN signaling and T cell recruiting cytokines, we performed an animal study using an immunogenic, PARPi-resistant model of ovarian cancer to determine if this dual therapy has any anti-tumor effects. Indeed, we observed that single and dual EHMTi therapy significantly reduced tumor burden and that this reduction was partially dependent on CD8 T cells. Lastly, to determine if dual therapy also induces a viral mimicry response in the human tumor microenvironment, we created ex vivo cultures of tumors resected from patients with ovarian cancer. We then treated the ex vivo tissues with DMSO, PARPi, EHMTi, and dual PARPi/EHMTi, and performed bulk RNA-sequencing and multispectral immunohistochemistry. From these analyses, we found that dual therapy upregulated TE transcripts and multiple immune-related pathways, and increased T cell activity in the tumor microenvironment. Altogether, our data suggest that dual therapy activates viral mimicry and promotes anti-tumor immunity in both PARPi-resistant and -sensitive ovarian cancer. Citation Format: Lily Nguyen, Zachary L. Watson, Elizabeth R. Woodruff, Kimberly R. Jordan, Edward Chuong, Benjamin G. Bitler. Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B009.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Clinicopathologic Overlap of Vulvar Psoriasis and Candidiasis.

The study's aim is to assess if vulvar psoriasis and candidiasis may be distinguished by clinical presentation and histopathologic appearance. The pathology database identified biopsies with corneal or subcorneal neutrophils, acanthosis, and dermal lymphocytic infiltrate. Exclusions were age younger than 18 years and unavailable or uninterpretable slides. Clinical data included demographics, comorbid conditions, symptoms, examination, microbiology, treatment, and response. Histopathologic review documented site, thickness, and characteristics of stratum corneum and epidermis, distribution of neutrophils, and infiltrate. Cases were stratified by microbiologic presence or absence of Candida albicans. Biopsies from 62 women with median age of 60 years were associated with C. albicans on vulvovaginal culture in 28 (45%), whereas 26 (42%) were negative, and 8 (13%) lacked microbiologic assessment. Swab-positive women were more likely to have diabetes, receive prereferral estrogen, and report vulvar pain. Specialist clinical impression was candidiasis in 33 (53%), psoriasis in 11 (18%), comorbid candidiasis and psoriasis in 7 (11%), dermatitis in 10 (16%), and unknown in 2 (3%). Visible fungal organisms occurred in 16 (26%) cases and were associated with diabetes and satellite lesions. Other than presence of organisms, there were no histopathologic differences stratified by microbiologic result. The histopathologic triad of corneal/subcorneal neutrophils, acanthosis, and dermal lymphocytic infiltrate is common to vulvar psoriasis and candidiasis, and clinical features do not reliably distinguish between them. Microbiologic assessment and single-agent treatment are useful strategies to clarify the diagnosis.

Comparison of Tepotinib, Paclitaxel, or Ramucirumab Efficacy According to the Copy Number or Phosphorylation Status of the MET Gene: Doublet Treatment versus Single Agent Treatment.

Although conventional combination chemotherapies for advanced gastric cancer (GC) increase survival, such therapies are associated with major adverse effects; more effective and less toxic treatments are required. Combinations of different anti-cancer drugs, for example, paclitaxel plus ramucirumab, have recently been used as second-line treatments for advanced GC. This study evaluated how copy number variations of the MET gene, MET mutations, and MET gene and protein expression levels in human GC cells modulate the susceptibility of such cells to single-agent (tepotinib, ramucirumab, or paclitaxel) and doublet (tepotinib-plus-paclitaxel or ramucirumab-plus-paclitaxel treatment regimens. Compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibited the growth of GC cells with MET exon 14 skipping mutations and those lacking MET amplification but containing phosphorylated MET; such inhibition was dose-dependent and associated with cell death. Tepotinib-plus-paclitaxel and ramucirumab-plus-paclitaxel similarly inhibited the growth of GC cells lacking MET amplification or MET phosphorylation, again in a dose-dependent manner, but without induction of cell death. However, tepotinib alone or tepotinib-plus-ramucirumab was more effective against c-MET-positive GC cells (>30 copy number variations) than was ramucirumab or paclitaxel alone or ramucirumab-plus-paclitaxel. These in vitro findings suggest that compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibits the growth of c-MET-positive GC cells, cells lacking MET amplification but containing phosphorylated MET, and cells containing MET mutations. Clinical studies are required to confirm the therapeutic effects of these regimens.

Abstract IA021: Digital patient avatars predict cancer evolution and personalised therapeutic strategies

Abstract Tumour heterogeneity manifested by genetic diversity and interchangeable cell states poses considerable challenges for the design of therapeutic strategies to combat cancer. While many strategies have been proposed to address this problem, using combination treatments and sequential delivery of therapies, the scope of combinations and sequencing order of therapies is enormous and is beyond the capacity of exhaustive testing through experimental models. We therefore propose using computational models to rapidly identify effective therapeutic interventions supported by convincing mechanistic explanations. Digital cancer avatars are computational programs representing oncogenic signalling networks and mapping molecular interactions to cell phenotype, which can be interrogated in silico. These computational programs specify how molecular interactions and perturbations produce cellular responses, such as cell proliferation, cell survival or cell death, and as such are well-suited to provide novel mechanistic insights into cellular behaviours as well as to identify therapeutic strategies for targeting them. In this talk, I will demonstrate the utility of bespoke computational models for triple-negative breast cancer, non-small cell lung cancer and melanoma to study how genomic changes drive tumour evolution and therapeutic response, and how these models are used to identify personalised combination treatments to overcome resistance. I will also present an in silico method applied to these models, to screen tens of millions combinations for effective sequential treatments by mapping the evolution of a tumour in response to a single-agent treatment and determining the optimal second-line treatment that will overcome resistance. Together these studies demonstrate the utility of digital avatars to improve our understanding of the mechanisms driving tumour progression and pave the way for personalised therapeutic strategies for improved cancer patient outcomes. Citation Format: Jasmin Fisher. Digital patient avatars predict cancer evolution and personalised therapeutic strategies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA021.

Adjuvant Chemotherapy and Outcomes in Older Adult Patients With Biliary Tract Cancer

The association of adjuvant chemotherapy (AC) with survival in the general population of patients with resected biliary tract cancer (BTC) remains controversial. As such, the role of this treatment in the treatment of older adult patients (aged ≥70 years) needs to be evaluated. To describe the patterns of use of AC and compare survival outcomes of AC and observation in older adult patients following resection of BTC. This retrospective cohort study included 8091 older adult patients with resected BTC with data available in the National Cancer Database from January 1, 2004, to December 31, 2019. Patients were divided into 2 cohorts: AC and observation. The AC cohort was subdivided into single-agent and multiagent AC treatment. Adjuvant chemotherapy vs observation following BTC resection. The primary outcome was overall survival (OS) of patients who received AC compared with observation following resection of BTC as evaluated using Kaplan-Meier estimates and multivariable Cox proportional hazards regression models. Inverse probability of treatment weighting and propensity score matching were performed to address indication bias. Between 2004 and 2019, of 8091 older adult patients with resected BTC identified (median [range] age, 77 [70-90] years; 5136 women [63.5%]; 2955 men [36.5%]), only one-third (2632 [32.5%]) received AC. There was an increase in the use of AC across the study period from 20.7% (n = 495) in 2004 to 2009 to 41.2% (n = 856) in 2016 to 2019. Age 80 years or older (odds ratio, 0.29; 95% CI, 0.25-0.33; P < .001) and gallbladder primary site (odds ratio, 0.71; 95% CI, 0.61-0.83; P < .001) were associated with a lower odds of AC. Following inverse probability of treatment weighting, as a composite, AC was not associated with improved survival (median OS, 20.5 months; 95% CI, 19.2-21.7 months) compared with observation (median OS, 19.0 months; 95% CI, 18.1-20.3 months). A longer median OS was associated with single-agent AC (21.5 months; 95% CI, 19.9-24.0 months) but not multiagent AC (19.1 months; 95% CI, 17.5-21.1 months) compared with observation (median OS, 17.3 months; 95% CI, 16.1-18.4 months). This improvement in OS with single-agent AC was not apparent on multivariable analysis (hazard ratio [HR], 0.97; 95% CI, 0.89-1.05; P = .44). However, age at diagnosis of 80 years or older (HR, 1.35; 95% CI, 1.28-1.42; P < .001) and treatment at nonacademic centers (HR, 1.14; 95% CI, 1.07-1.20, P < .001) were associated with worse OS. In this cohort study of older adult patients, AC was not associated with an improvement in survival compared with observation following BTC resection. These findings suggest the need for further study of AC for older adult patients who may benefit after curative intent surgery for BTC.

Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

Aberrant activation of the PI3K/AKT signaling axis along with the sustained phosphorylation of downstream BAD is associated with a poor outcome of TNBC. Herein, the phosphorylated to non-phosphorylated ratio of BAD, an effector of PI3K/AKT promoting cell survival, was observed to be correlated with worse clinicopathologic indicators of outcome, including higher grade, higher proliferative index and lymph node metastasis. The structural optimization of a previously reported inhibitor of BAD-Ser99 phosphorylation was therefore achieved to generate a small molecule inhibiting the phosphorylation of BAD at Ser99 with enhanced potency and improved oral bioavailability. The molecule 2-((4-(2,3-dichlorophenyl)piperazin-1-yl)(pyridin-3-yl)methyl) phenol (NCK) displayed no toxicity at supra-therapeutic doses and was therefore assessed for utility in TNBC. NCK promoted apoptosis and G0/G1 cell cycle arrest of TNBC cell lines in vitro, concordant with gene expression analyses, and reduced in vivo xenograft growth and metastatic burden, demonstrating efficacy as a single agent. Additionally, combinatorial oncology compound library screening demonstrated that NCK synergized with tyrosine kinase inhibitors (TKIs), specifically OSI-930 or Crizotinib in reducing cell viability and promoting apoptosis of TNBC cells. The synergistic effects of NCK and TKIs were also observed in vivo with complete regression of a percentage of TNBC cell line derived xenografts and prevention of metastatic spread. In patient-derived TNBC xenograft models, NCK prolonged survival times of host animals, and in combination with TKIs generated superior survival outcomes to single agent treatment. Hence, this study provides proof of concept to further develop rational and mechanistic based therapeutic strategies to ameliorate the outcome of TNBC.