Trials For Solid Tumors Research Articles

Abstract 6206: Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor. Despite high initial responses to frontline chemo-immunotherapy, therapeutic resistance develops rapidly. There are limited treatment options in the relapsed setting, where the prognosis remains dismal. SCLC tumors experience continuous and high levels of replication stress (RS) due to ubiquitous loss of key cell cycle checkpoints, RB1 and TP53. Frequent amplification and high expression of the transcription factor cMYC further contribute to increased RS. Thus, high levels of RS expose a potential SCLC vulnerability and provide a therapeutic opportunity. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its role in driving epithelial to mesenchymal transition (EMT). More recently, a novel role for AXL in DNA damage repair and tolerance has emerged. Therefore, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We first investigated the transcriptomic expression profile of AXL in SCLC clinical cohorts. AXL-high tumors were seen in a subset of treatment-naïve SCLC tumors, frequently among, but not limited to, the inflamed SCLC subtype. AXL expression was also seen in many relapsed SCLC tumors. As expected, tumors with high AXL expression also expressed several mesenchymal genes and higher EMT scores. Interestingly, among the treatment-naïve SCLC tumors, AXL expression was inversely correlated with a RS signature (rho=-0.54, p<0.001). Next, we tested the effects of AXL inhibition in SCLC in vitro and in vivo models. In a panel of 30 SCLC cell lines, bemcentinib, a selective AXL inhibitor in clinical trials for various advanced solid tumors, exhibited a range of antiproliferative activity, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM). Bemcentinib also significantly delayed tumor growth in in vivo SCLC models. Biomarkers associated with sensitivity to bemcentinib in SCLC cell lines included markers of RS (cMYC, replication stress score) and DNA damage response (phospho CHK1S345, phospho CHK2T68). Bemcentinib also induced RS, indicated by the activation of ATR/CHK1-mediated RS response pathway, and DNA damage, and the combination with an ATR inhibitor (ceralasertib) showed a greater than additive effect. In a syngeneic model of SCLC, the combination of bemcentinib, ceralasertib and an anti-PDL1 antibody induced significant tumor regression. Together, these promising findings demonstrate that AXL inhibition may be an effective strategy to target the RS vulnerability common in SCLC. Citation Format: Kavya Ramkumar, C. Allison Stewart, Azusa Tanimoto, Qi Wang, Yuanxin Xi, Benjamin B. Morris, Runsheng Wang, Li Shen, Robert J. Cardnell, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6206.

Abstract 4044: Development of UCT-01-097, a novel orally available ERK1/2 inhibitor for the treatment of ERK1/2 dependent cancers

Abstract The MAPK signaling pathway is the most commonly mutated and/or dysregulated pathway in cancer. Strategies to target it have yielded some success with inhibitors against KRASG12C and BRAF, and to a lesser extent, MEK1/2. However, the impact of these molecules is often limited by toxicity and rapid and diverse mechanisms of resistance; both adaptive and/or acquired. For example, treatment with MAPK pathway targeting agents results in compensatory activation of the downstream mediator ERK1/2 and enables tumors to subvert the targeted therapy. Thus, targeting ERK1/2 provides promising potential advantages in overcoming and/or preventing adaptive and acquired resistance. We evaluated multiple preclinical and clinically staged ERK1/2 inhibitors—including ERAS-007 and BVD-523—in a 500+ cell line screening platform and identified cancers with subpopulations that are sensitive to this class of inhibitor. KiNativ analyses helped to inform the differences in sensitivity/selectivity that we observed between each ERK1/2 inhibitor. Comprehensive molecular profiling of the cell lines at baseline allowed us to screen for potential molecular markers of sensitivity/resistance to these compounds. Using this platform, we have developed a novel, potent ERK1/2 small molecule inhibitor, UCT-01-097, with improved selectivity over other clinically staged inhibitors. These data, coupled with the broad spectrum of in vitro responses, suggests an improved therapeutic index with this molecule. UCT-01-097 shows kinase selectivity in both cell free and in-cell assays and robust efficacy in panel of pancreatic PDX models. Inhibition of xenograft tumor growth was achieved using both daily dosing and intermittent dosing schedules. We have successfully submitted a regulatory IND and are currently enrolling a Phase 1 clinical trial in advanced solid tumors for treatment with UCT-01-097 (NCT04761601). Citation Format: Neil A. O'Brien, Martina S. McDermott, Brendan M. O'Boyle, Corey M. Reeves, Michael Bartberger, Oliver Loson, Kevin Chau, Jenny J. Hong, Weiping Jia, Naeimeh Kamranpour, Tong Luo, Raul Ayala, Athena M. Madrid, John A. Glaspy, Brian M. Stoltz, Dennis J. Slamon. Development of UCT-01-097, a novel orally available ERK1/2 inhibitor for the treatment of ERK1/2 dependent cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4044.

Abstract 6368: Preclinical pharmacology and safety studies of ZG005: an anti-PD-1/TIGIT bispecific mAb in a phase I clinical trial for advanced tumors

Abstract ZG005 is a humanized anti-PD-1/TIGIT bispecific antibody that effectively blocks the binding between PD-1 and TIGIT, on immune cells, with their specific ligands PD-L1 and PVR, on tumors. As a dual blockade of PD-1/TIGIT immune checkpoints, ZG005 exhibits sustained occupancy to both targets and inhibits their pathways specifically and simultaneously, leading to synergistic effects and boosting the ability of the immune system in killing tumor cells. With high binding affinities, ZG005 elicits the activation of T cells and NK cells resulting in increased release of cytokines and the depletion of Tregs, independent of ADCC activities. ZG005 utilizes the IgG4 isotype to avoid the potential negative impact of effector function and target-crosslinking that has been reported for anti-TIGIT therapeutics using IgG1 isotype. In vivo animal studies indicate that ZG005 has potent and dose-dependent anti-tumor efficacies in syngeneic models using CT26 and MC38 colon tumors in humanized PD-1/TIGIT dKIs mice. In these models, the whole blood and TIL analyses confirmed increases in CD8+ and NK cells but reduction of Treg cell levels after ZG005 treatments. The combination of ZG005 with chemotherapeutic-reagents, cisplatin and donafenib, enhanced their anti-tumor efficacies. Toxicity and safety pharmacology studies with weekly repeat doses for four weeks in cynomolgus monkeys, indicate that ZG005 is well tolerated. Over the dose range 20-180 mg/kg, there were no obvious toxic or adverse effects, and the HNSTD and NOAEL values were 180 mg/kg and 60 mg/kg, respectively. PK/TK analyses indicate a prolonged ZG005 receptor occupancy over 80%, consistent with in vitro binding results that are attribute to the S228P mutation in the IgG4 hinge region to prevent Fab exchange and enhance stability. The IND application of ZG005 has been approved by both FDA and NMPA, and the molecule is currently in phase I clinical trials for advanced solid tumors at escalated dosing of 0.3~20 mg/kg, Q3W, via i.v. administration. Citation Format: Bing Zhu, Tongcheng Dai, Ruifeng Liu, Alfonso Suarez, Tyler Liban, Bin Zhang, Margaret Karow, Jackie Sheng, Zelin Sheng, Binhua Lv. Preclinical pharmacology and safety studies of ZG005: an anti-PD-1/TIGIT bispecific mAb in a phase I clinical trial for advanced tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6368.

Abstract 6247: Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response

Abstract Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (<15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (<15%, N=9) vs. 100.0% (≥15%, N=6) p<0.0001). Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6247.

Abstract 2561: Computational drug repositioning identifies Akt inhibitor MK2206 binding to EZH2 as enhancer of Tazmetostat efficacy in inhibition of triple negative breast cancer proliferation

Abstract The histone methyltransferase Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). Its overexpression in triple-negative breast cancer (TNBC) correlates with aggressive, poorly differentiated phenotypes and poor prognosis. EZH2 methylates histone 3 at lysine 9/27 and controls the transcription of many genes, such as cyclin D1, Cyclin E and MMP9, which are involved in tumor survival, proliferation, migration, invasion, and angiogenesis. EZH2 also promotes breast tumor-initiating cell expansion (BITC) through impairment of the DNA repair process and the favoring of oncogenes. Small-molecule EZH2 inhibitors, including Tazmetostat (TAZVERIK), mainly inhibit the SET domain methylase activity which may not completely abrogate the actions of EZH2. Recently, TAZVERIK was FDA approved for follicular lymphoma and is in clinical trials for other solid tumors including the breast. The PI3K-AKT pathway in TNBC plays a pivotal role in metastasis and tumorigenesis, and it associates and interacts with other proteins and pathways. Post translational modifications of EZH2 lead to its role in cancer progression, and importantly, AKT phosphorylates EZH2 and promotes oncogenesis independent of PRC2-mediated gene transcriptional silencing. Docking studies using PyRx 0.8 software revealed AKT inhibitor MK2206 interact with EZH2. We hypothesize that enhancing EZH2 inhibition by combining Tazmetostat with an Akt inhibitor will be highly effective in suppressing progression and metastasis in TNBC. The in-silico analysis revealed MK2206 actively interacts with the CXC (Cystein rich region) an integral part of the catalytic site of EZH2. The binding affinity of MK2206 was observed to be -6.9 kcal/mol, which is -1.3 kcal/mol less than the affinity of Tazmetostat (-8.2Kcal/mol). Tazmetostat binds to the SET domain, inhibiting methyltransferase activity, while MK2206 binds towards the CXC region. Tazmetostat by itself effectively inhibit cell growth (IC50: 20.3 μM) but combining with MK2206 caused a synergistic inhibition of cell viability. The combination of 8µM Tazmetostat and 5μM MK2206 significantly suppressed EZH2 activity and migration. Additionally, we investigated the Protein-Protein interaction STRING between AKT and EZH2 in humans, which revealed interactions with proteins involved in regulation of cell cycle progression (CDK2), RING -type E3 Ubiquitin Transferase (TTC3), and the triggering of histone H3-k27 methylation (EED). The combination of Tazmetostat and MK2206 significantly inhibits TNBC proliferation, invasion and migration by disrupting the catalytic activity by preventing EZH2-DNA interactions, abrogating methylation and inhibiting the EZH2-Akt interaction in the TNBC phenotype. (Work supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Gowripriya Thirumugam, Gautham Sarathy, Shiv Verma, Shivani Dhekne, Balamurugan Krishnaswamy, Sanjay Gupta, Bhuvaneswari Ramaswamy, Eswar Shankar. Computational drug repositioning identifies Akt inhibitor MK2206 binding to EZH2 as enhancer of Tazmetostat efficacy in inhibition of triple negative breast cancer proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2561.

Abstract 3118: Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks

Abstract Introduction: PARP inhibitors (PARPi) are known to have a synthetic lethal (SL) relationship with BRCA1/2 mutated cancers (ie: BRCA loss confers sensitivity to PARPi), but heterogeneity in response exists. PARPi have also found utility in BRCA-non mutated cancers, suggesting other factors may contribute to response. Recently, PARPi trials in pediatric solid tumors have had mixed results. SL interactions have been shown to exist in a network relationship, as such the aim of this study was to identify a clinically relevant SL signature (including synthetic dosage lethality and synthetic rescue) that would predict response to PARPi in pediatric cancers. Methods: Pre-treatment genomic copy number alteration, RNA sequencing, and survival data from 519 pediatric cancer patients (INFORM Consortium, 91% solid tumor) were obtained. SL partner genes for PARPi were selected if co-alteration of the drug target (PARP1/2/3) and candidate gene was 1) found to be under-represented in the patient transcriptome, 2) associated with improved survival, and 3) the genes were phylogenetically similar to PARP1/2/3. The 100 gene signature was validated on two independent pediatric cancer drug screens: in-vivo orthotopic patient-derived xenograft (OPDX) mouse models (n=24) and in-vitro PRISM cell lines (n=54). It was also validated on a cisplatin (PARP surrogate) treated pediatric osteosarcoma clinical trial (n=37). Comparisons were made to 1) a signature generated with the same pipeline using adult TCGA data, 2) a validated adult synthetic lethality pipeline (SELECT), and 3) a published 60-gene PARPi response signature (BRCAness score). Results: The pediatric signature predicted response in all drug screens evaluated. Four cell line screens: Talazoparib (AUC 0.72, p = 0.003), Rucaparib (AUC 0.69, p = 0.01), Niraparib (AUC 0.67, p = 0.03), Olaparib (AUC 0.64, p = 0.06), and two OPDX screens: Talazoparib (AUC 0.74, p = 0.03), Olaparib (AUC 0.74, p = 0.03). The pediatric signature outperformed both adult signatures (AUCs 0.34-0.61, all p’s >0.05), and the BRCAness score (AUCs 0.41-0.53, p’s >0.05) except in the Olaparib cell line (BRCAness: AUC 0.68, p = 0.01). In the pediatric osteosarcoma dataset, the pediatric signature predicted death (AUC 0.68) and two-year recurrence (AUC 0.72), outperforming the other measures (death: AUC 0.49-0.66; two-year recurrence: AUCs 0.36-0.44). Discussion: We developed a pediatric derived, clinically relevant, synthetic lethal signature that predicts response to PARPi in pan-pediatric cancer drug screens and in a cisplatin treated osteosarcoma clinical trial. Our findings support the goal of identifying precision oncology frameworks to improve the care of children with cancer. Citation Format: Matthew Nagy, Fiorella Schischlik, Kun Wang, Nishanth Ulhas Nair, E. Michael Gertz, Lipika R. Pal, Natalie Jaeger, Christopher Previti, Dina ElHarouni, Stefan M. Pfister, Eytan Ruppin. Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3118.

Abstract 3247: Combination therapy using PERK and PD1/PD-L1 inhibitors reprograms tumor associated macrophages and reduces tumor burden

Abstract Background: The role of endoplasmic reticulum (ER) stress is profound in increasing malignant activity of neoplastic tumor cells and has been recently demonstrated to also increase immunosuppressive activity of tumor-associated macrophages (TAMs). Immunosuppressive TAM activity is thought to underly resistance to PD-1/PD-L1-targeted immune checkpoint inhibitors used for cancer therapy. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is a rate-limiting enzyme central to the ER-stress response mechanism that transduces ER-stress signals in cells to regulate protein synthesis, cell death, and cell survival. We hypothesized that inhibition of PERK would improve responses to ICI therapy by reprogramming TAM from immunosuppressive to immunoactivating cells. Methods: To investigate if ER-stress underlies resistance to PD-1/PD-L1 targeted therapies, we utilized ex vivo assays to investigate ER-stress regulation of macrophage phenotype, and in vivo syngeneic murine models of melanoma growth, with HC-5404 (PERKi), a selective and potent first-in-human small molecule PERK inhibitor currently in a phase 1 clinical trial for solid tumors (NCT04834778). Results: Treatment with PERKi sensitized αPD-1/PD-L1 mAb-resistant melanoma tumors (Y1.7/YR1.7) to PD-1/PD-L1 blockade with concomitant increase in tumor infiltrating leukocytes, TH1- reprogrammed TAM, and cytotoxic CD8+ T cells. In addition to tumor microenvironment changes by PERKi, gene expression analysis confirmed reprograming of the tumor microenvironment, marked by a relative increase in TH1-associated genes and downregulation of TH2-associated genes when compared with untreated tumors. We utilized an ex vivo macrophage-splenocyte co-culture assay to depict macrophage dependent T cell suppression prevalent in tumors. This revealed that treating macrophages with PERKi prior to co-culturing with T cells or adding PERKi to the macrophage-T cell co-culture relieved macrophage dependent T cell suppression, illustrating a potential mechanism of therapeutic efficacy. We are now studying how ER-stress is regulated in TAMs and are focusing on identifying components of PERK-dependent cell signaling that increases the tumor promoting behavior of TAMs. Conclusions: The combination therapy targeting PERK and PD-1/PD-L1 signaling increased adaptive immune responses, reprogramed TAMs and reduced tumor growth kinetics. Results from these studies highlight a role for ER-stress signaling in TAMs to maintain an immunosuppressive tumor microenvironment and demonstrate the potential therapeutic strategy of PERK inhibition in αPD-1/PD-L1 resistant tumors. Citation Format: Sushil Kumar, Michael McCane, Jeremy Drees, Nandita Bose, Lisa Coussens. Combination therapy using PERK and PD1/PD-L1 inhibitors reprograms tumor associated macrophages and reduces tumor burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3247.

Abstract 5018: PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation

Abstract CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with their binding partner cyclin C (CCNC) and two other proteins; the CDK module is associated with transcriptional Mediator complex. CDK8/19 Mediator kinases potentiate transcription induced by different signals, regulate protein levels of Mediator complex components and protect CCNC from proteolytic degradation; the latter activity is exerted in a kinase-independent manner. CDK8/19 kinase inhibitors have entered clinical trials for solid tumors and leukemias. To extend the effects of CDK8/19 inhibition and to suppress their kinase-independent activities, we have developed three series of PROteolysis TArgeting Chimeras (PROTACs) based on different selective inhibitors of CDK8/19 kinases, connected via different linkers to a moiety binding cereblon (CRBN) E3 ligase. The most potent PROTACs degraded both CDK8 and CDK19 in different cell types with DC50s of 10-20 nM. RNA-Seq analysis of 293 cells treated with a CDK8/19 kinase inhibitor or PROTAC showed that the bulk of the PROTAC’s transcriptomic effects matched the effects of the kinase inhibitor. In contrast to the kinase inhibitors, CDK8/19-degrading PROTACs also induced CCNC degradation. Cancer Dependency Map (DepMap) analysis revealed that multiple myelomas (MM), where CRBN is an established therapeutic target, show greater dependency on CCNC than most of the tumor cell lines. We have tested two strongly CCNC-dependent MM cell lines and one CCNC-independent MM line for sensitivity to different CDK8/19 kinase inhibitors and PROTACs. Kinase inhibitors showed low to moderate anti-proliferative activity in all three MM lines, whereas CDK8/19 PROTACs were an order of magnitude more potent than the kinase inhibitors in the two CCNC-dependent MM lines (IC50s of 20-30 nM) but not in the CCNC-independent line. The effects of CDK8/19-degrading PROTACs were stronger than those of the CRBN-targeting drug pomalidomide (approved for MM), a CRBN-binding PROTAC acting on androgen receptor or a CDK8 degradation-inactive PROTAC analog containing CDK8/19- and CRBN-binding moieties. These results suggest the potential of CDK8/CDK19-degrading PROTACs for the treatment of MM. Citation Format: Li Zhang, Jing Li, Charles E. Dowling, Eugenia V. Broude, Igor B. Roninson, Campbell McInnes, Mengqian Chen. PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5018.

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer.

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

Reactivated thyroid hormone receptor β attenuates anaplastic thyroid cancer (ATC) stem cell activity.

Anaplastic thyroid cancer (ATC) is one of the most aggressive solid cancers in humans, with limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity contributes to therapeutic resistance and recurrence of ATC. We show that the expression of the endogenous thyroid hormone receptor β gene (THRB) is silenced in ATC and demonstrate that the exogenously expressed TRβ suppresses CSC activity. Decitabine is one of the demethylation agents to treat myelodysplastic syndrome and acute myeloid leukemia patients and is currently in clinical trials for hematopoietic malignancies and solid tumors. We aim to show that the re-expression of the endogenous THRB gene by decitabine can attenuate CSC activity to block ATC tumor growth. We treated ATC cell lines derived from human ATC tumors (11T and 16T cells) with decitabine and evaluated the effects of the reactivated endogenous TRβ on CSC activity in vitro and in vivo xenograft models. We found that treatment of 11T and 16T cells with decitabine reactivated the expression of endogenous TRβ, as evidenced by western blot and immunohistochemical analyses. The expressed TRβ inhibited cell proliferation by arresting cells at the S phase, increased apoptotic cell death by upregulation of cleaved caspase-3, and markedly suppressed the expression of CSC regulators, including cMYC, ALDH, SOX2, CD44, and β-catenin. Decitabine also inhibited xenograft tumor growth by suppressing CSC activity, inhibiting cancer cell proliferation, and increasing apoptosis. Our findings suggest that re-expression of the endogenous TRβ is a novel therapeutic approach for ATC via suppression of CSC activity.

Co-enrichment of CD8-positive T cells and macrophages is associated with clinical benefit of tislelizumab in solid tumors

BackgroundActivated immune cells (IC) in the tumor microenvironment (TME) are critical for anti-tumor efficacy. Greater understanding of the dynamic diversity and crosstalk between IC is needed to clarify their association with immune checkpoint inhibitor efficacy.MethodsPatients from three tislelizumab monotherapy trials in solid tumors (NCT02407990, NCT04068519, NCT04004221) were retrospectively divided into subgroups by CD8+ T-cell and macrophage (Mφ) levels, assessed via multiplex immunohistochemistry (mIHC; n = 67) or gene expression profiling (GEP; n = 629).ResultsA trend of longer survival was observed in patients with both high CD8+ T-cell and Mφ levels versus other subgroups in the mIHC analysis (P = 0.11), which was confirmed with greater statistical significance in the GEP analysis (P = 0.0001). Co-existence of CD8+ T cells and Mφ was coupled with elevated CD8+ T-cell cytotoxicity, T-cell trafficking, MHC class I antigen presentation signatures/genes, and enrichment of the pro-inflammatory Mφ polarization pathway. Additionally, a high level of pro-inflammatory CD64+ Mφ density was associated with an immune-activated TME and survival benefit with tislelizumab (15.2 vs. 5.9 months for low density; P = 0.042). Spatial proximity analysis revealed that closer proximity between CD8+ T cells and CD64+ Mφ was associated with a survival benefit with tislelizumab (15.2 vs. 5.3 months for low proximity; P = 0.024).ConclusionsThese findings support the potential role of crosstalk between pro-inflammatory Mφ and cytotoxic T cells in the clinical benefit of tislelizumab.Trial registrationNCT02407990, NCT04068519, NCT04004221.

Investigating documented reasons for withdrawal from clinical trials for patients with advanced prostate cancer (PCa).

105 Background: In clinical trials, treatment efficacy rates are based on participants who can tolerate treatment through study endpoints. Attrition from clinical trials can potentially lead to biased results (PMID: 30379822). Many oncology clinical trials censor the subject data of participants because of early trial intervention discontinuation due to adverse events (AEs), withdrawal of consent, or loss to follow-up. Despite high attrition rates reported across various oncology clinical trials (10-38%), there is inconsistency regarding investigating the reasons for withdrawal (PMID: 23132290). We aim to identify the causes of the withdrawal of participants with advanced PCa from clinical trials. Methods: In this IRB-approved single-center retrospective study (Sept 2014 to Sept 2022), the OnCore Research Database was used to capture data on the participants with advanced PCa enrolled in clinical trials. The included trials were specific to prostate cancer, and mixed solid tumor trials were excluded. Endpoint reports from all included trials were assessed. Participants who discontinued treatment for any reason were included. The primary objective was to obtain reasons for clinical trial withdrawal before primary endpoints, identify potential clinical intervention areas, and determine potential areas of adjustment to future study designs to improve retention rates. Eligibility criteria: diagnosis of metastatic prostate cancer, receipt of protocol treatment until discontinuation, and documentation of a reason for discontinuation of the protocol treatment. Results: 272 pts from the 18 clinical trials met eligibility criteria and were included in the study. The most common reasons documented for discontinuation were: the progression of disease (46.69%), other reasons (15.44%), adverse events (5.15%), personal preference (15.81%), and completion of protocol treatment (11.76%) of the total number of enrolled participants. We noted a statistically significant difference between participants who withdrew for other reasons in the castration-resistant versus castration-sensitive setting (6.08% vs. 32.38%, p value=0.03). Notably, declining performance status and symptom progression were listed under the “other reasons” category. The documentation of patient withdrawal due to clinical symptoms was reported across multiple withdrawal categories. Conclusions: In approximately one-third of participants, the reason for protocol treatment discontinuation was not apparent (documented as patient preference and other reasons with no further specifications). Further exploration of the exact reasons contributing to therapy discontinuation in these patients will be critical to improving patient retention in clinical trials.

Targeting Transcription Factors ATF5, CEBPB and CEBPD with Cell-Penetrating Peptides to Treat Brain and Other Cancers.

Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies.

Trial in progress: Natural killer (NK) cells with TGFβ receptor I inhibitor vactosertib and IL-2 in patients with metastatic colorectal cancer or hematologic malignancies.

TPS273 Background: Natural killer (NK) cell therapy is emerging as a promising therapy for patients (pts) with solid tumors and hematologic malignancies with no reported toxicity associated with the cell therapy product itself. NK cell trials in solid tumors are limited by immune inhibitory tumor microenvironments that prevent trafficking and activity of donor NK cells, which is largely governed by TGFβ signaling. We recently developed a novel platform for ex vivo expansion of healthy donor NK cells. We previously showed these cells to be safe in a dose-escalation study when given without cytokine support, with early evidence of response in pts with metastatic colorectal cancer (CRC) and in pts with hematologic malignancies. IL-2 has been well-established as an important activating cytokine supporting NK cells in humans, but can also induce regulatory T cells, which further suppress NK cells by producing TGFβ. We seek to improve upon the early activity we previously observed with NK cells alone by administering concurrently with IL-2 and by limiting the inhibitory tumor microenvironment and regulatory T cells with TGFβ receptor 1 inhibitor vactosertib. Methods: This phase Ib open-label study evaluates the safety and persistence (primary objectives), and the clinical and biological activity and trafficking (secondary objectives) of our novel NK cell therapy in combination with IL-2 (aldesleukin) and TGFβ receptor 1 inhibitor vactosertib. Up to 12 pts with metastatic CRC or relapsed/refractory hematologic malignancies will be enrolled. Pts will undergo low-dose lymphodepletion with fludarabine and cyclophosphamide followed by 2 infusions of NK cells, administered 14 days apart. Concurrently, pts will receive aldesleukin subcutaneously 3 times weekly and vactosertib orally 5 consecutive days/week x4 weeks total. Pts will undergo pre- and post-treatment biopsies for quantification of NK cell trafficking and exploration of immune cell profiling changes in the tumor microenvironment. The trial is currently open to enrollment, with 1 pt accrued at time of submission. Clinical trial information: NCT05400122 .

A phase I clinical trial of stereotactic body radiotherapy with atezolizumab and bevacizumab in advanced hepatocellular carcinoma.

TPS626 Background: Systemic therapy with atezolizumab and bevacizumab (atezo/bev) has improved outcomes for advanced HCC, but results in objective responses in fewer than 30% of patients. Stereotactic body radiotherapy (SBRT) is currently used for small HCC tumors that do not require systemic therapy but has also been shown in a number of clinical trials of other solid tumors to enhance the anti-cancer immune response. Of particular interest, our prior experience with SBRT in a 17Gy fraction has demonstrated the ability to restore sensitivity to immunotherapy in advanced solid tumors, even those previously refractory to immunotherapy. We hypothesize that repeated high dose fractions of radiation will act as an immune booster and will improve on outcomes of patients with advanced HCC. Since the combination of SBRT and atezo/bev has not yet been tested prospectively in patients with HCC, and because bevacizumab is a known radiosensitizer, we are conducting a phase I trial to evaluate the safety of repeated SBRT doses. Methods: This is a single-site phase I clinical trial utilizing a Rolling 6 design to determine the safety of 1, 2, or 3 doses of SBRT fractions in combination with atezo/bev. Up to 18 total patients will be enrolled (n=6 per cohort). Patients must be naïve to systemic therapy with Child-Pugh A or B liver function, at least one lesion amenable to radiation, and a measurable lesion that will not receive radiation. Patients with uncontrolled ascites or hepatic encephalopathy are excluded. Atezo/bev is administered at the standard doses every 21 days. SBRT will start 1 week after the first infusion of this combination. Patients will receive 1, 2, or 3 17Gy fractions of SBRT at 4-week intervals (cohorts 2 and 3 only). Patients will undergo serial collections of circulating cell-free DNA (ccfDNA), methylated DNA, and peripheral blood mononuclear cells to investigate the application of these markers as a predictor of response. Following completion of SBRT, patients will continue with atezo/bev until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint is the proportion of patients experiencing dose-limiting toxicities (grade 3 or higher radiation-related toxicities graded by the Common Terminology Criteria for Adverse Events, Version 5). Secondary endpoints are overall survival, progression-free survival, objective response rate, duration of response, and toxicity rates. Enrollment began 9/2022 and complete accrual is expected by June 2024. NCT05488522. Clinical trial information: NCT05488522 .

PD-1 blockade augments CD8+ T cell dependent antitumor immunity triggered by Ad-SGE-REIC in Egfr-mutant lung cancer

ObjectivesNo immunotherapeutic protocol has yet been established in never-smoking patients with lung cancer harboring driver oncogenic mutations, such as epidermal growth factor receptor (EGFR) mutations. The immunostimulatory effect of Ad-REIC, a genetically engineered adenovirus vector expressing a tumor suppressor gene, reduced expression in immortalized cells (REIC), has been investigated in clinical trials for various solid tumors. However, the immunostimulatory effect of the Ad-REIC in EGFR-mutant lung cancer with a non-inflamed tumor microenvironment (TME) has not been explored. Materials and methodsWe used a syngeneic mouse model developed by transplanting Egfr-mutant lung cancer cells into single or double flanks of C57BL/6J mice. Ad-SGE-REIC, a 2nd-generation vector with an enhancer sequence, was injected only into the tumors from one flank, and its antitumor effects were assessed. Tumor-infiltrating cells were evaluated using immunohistochemistry or flow cytometry. The synergistic effects of Ad-SGE-REIC and PD-1 blockade were also examined. ResultsInjection of Ad-SGE-REIC into one side of the tumor induced not only a local antitumor effect but also a bystander abscopal effect in the non-injected tumor, located on the other flank. The number of PD-1+CD8+ T cells increased in both injected and non-injected tumors. PD-1 blockade augmented the local and abscopal antitumor effects of Ad-SGE-REIC by increasing the number of CD8+ T cells in the TME of Egfr-mutant tumors. Depletion of CD8+ cells reverted the antitumor effect, suggesting they contribute to antitumor immunity. ConclusionAd-SGE-REIC induced systemic antitumor immunity by modifying the TME status from non-inflamed to inflamed, with infiltration of CD8+ T cells. Additionally, in Egfr-mutant lung cancer, this effect was enhanced by PD-1 blockade. These findings pave the way to establish a novel combined immunotherapy strategy with Ad-SGE-REIC and anti-PD-1 antibody for lung cancer with a non-inflamed TME.

Bright future or blind alley? CAR-T cell therapy for solid tumors.

Chimeric antigen receptor (CAR) T cells therapy has emerged as a significant breakthrough in adoptive immunotherapy for hematological malignancies with FDA approval. However, the application of CAR-T cell therapy in solid tumors remains challenging, mostly due to lack of suitable CAR-T target antigens, insufficient trafficking and extravasation to tumor sites, and limited CAR-T survival in the hostile tumor microenvironment (TME). Herein, we reviewed the development of CARs and the clinical trials in solid tumors. Meanwhile, a "key-and-lock" relationship was used to describe the recognition of tumor antigen via CAR T cells. Some strategies, including dual-targets and receptor system switches or filter, have been explored to help CAR T cells matching targets specifically and to minimize on-target/off-tumor toxicities in normal tissues. Furthermore, the complex TME restricts CAT T cells activity through dense extracellular matrix, suppressive immune cells and cytokines. Recent innovations in engineered CARs to shield the inhibitory signaling molecules were also discussed, which efficiently promote CAR T functions in terms of expansion and survival to overcome the hurdles in the TME of solid tumors.

Sitravatinib as a potent FLT3 inhibitor can overcome gilteritinib resistance in acute myeloid leukemia

BackgroundGilteritinib is the only drug approved as monotherapy for acute myeloid leukemia (AML) patients harboring FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation throughout the world. However, drug resistance inevitably develops in clinical. Sitravatinib is a multi-kinase inhibitor under evaluation in clinical trials of various solid tumors. In this study, we explored the antitumor activity of sitravatinib against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.MethodsGrowth inhibitory assays were performed in AML cell lines and BaF3 cells expressing various FLT3 mutants to evaluate the antitumor activity of sitravatinib in vitro. Immunoblotting was used to examine the activity of FLT3 and its downstream pathways. Molecular docking was performed to predict the binding sites of FLT3 to sitravatinib. The survival benefit of sitravatinib in vivo was assessed in MOLM13 xenograft mouse models and mouse models of transformed BaF3 cells harboring different FLT3 mutants. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of sitravatinib.ResultsSitravatinib inhibited cell proliferation, induced cell cycle arrest and apoptosis in FLT3-ITD AML cell lines. In vivo studies showed that sitravatinib exhibited a better therapeutic effect than gilteritinib in MOLM13 xenograft model and BaF3-FLT3-ITD model. Unlike gilteritinib, the predicted binding sites of sitravatinib to FLT3 did not include F691 residue. Sitravatinib displayed a potent inhibitory effect on FLT3-ITD-F691L mutation which conferred resistance to gilteritinib and all other FLT3 inhibitors available, both in vitro and in vivo. Compared with gilteritinib, sitravatinib retained effective activity against FLT3 mutation in the presence of cytokines through the more potent and steady inhibition of p-ERK and p-AKT. Furthermore, patient blasts harboring FLT3-ITD were more sensitive to sitravatinib than to gilteritinib in vitro and in the PDX model.ConclusionsOur study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

Order of patient entry and outcomes in phase II clinical trials: A meta-analysis of individual patient data

BackgroundPrior meta-analysis of stem-cell transplantation trials for renal-cell carcinoma observed that clinical outcomes vary by subjects' order of entry, specifically their quartile of accrual. We test this hypothesis using meta-analysis of individual patient data from diverse Phase II trials conducted by an oncology consortium. MethodsEligible were all Phase II trials in hematologic or solid tumors opened and closed by California Cancer Consortium during 2005–2020. Excluded were trials closed in first quartile or currently embargoed pending publication and subjects ineligible per protocol or untreated on study. The primary risk factor was entry by quartile of planned sample size. As a cross-protocol endpoint, primary outcome was time to discontinuation of intervention. One-stage meta-analysis used a shared frailty model with trial as random effect. As covariates, stepwise selection retained tumor type, obesity, their interaction, calendar year, entry at least 3 years post-diagnosis, and performance status but rejected age, sex, randomized design, and class of drug. ResultsTwenty trials (including 8 terminated early, 2 not published) included n = 923 subjects. Most (90.6%) subjects discontinued intervention, usually for disease progression or toxicity. Independently of covariates, risk of discontinuation increased (p < 0.0001) with each quartile of entry (Hazards Ratio 1.13, 95% CI 1.06–1.22), culminating at Quartile 4 (HR 1.46, 1.36–1.57). The 95% prediction interval for the Hazards Ratio in future trials was (1.04–1.24). Progression-free survival similarly worsened by quartile of entry. ConclusionIn Phase II trials, clinical outcome worsens with quartile of entry. This finding merits independent replication, and the cause of this phenomenon merits investigation.

The natural killer cell immunotherapy platform: An overview of the landscape of clinical trials in liquid and solid tumors

The translation of natural killer (NK) cells to the treatment of malignant disease has made significant progress in the last few decades. With a variety of available sources and improvements in both in vitro and in vivo NK cell expansion, the NK cell immunotherapy platform has come into its own. The enormous effort continues to further optimize this platform, including ways to enhance NK cell persistence, trafficking to the tumor microenvironment, and cytotoxicity. As this effort bears fruit, it is translated into a plethora of clinical trials in patients with advanced malignancies. The adoptive transfer of NK cells, either as a standalone therapy or in combination with other immunotherapies, has been applied for the treatment of both liquid and solid tumors, with numerous early-phase trials showing promising results. This review aims to summarize the key advantages of NK cell immunotherapy, highlight several of the current approaches being taken for its optimization, and give an overview of the landscape of clinical trials translating this platform into clinic.