Adaptive Resistance Mechanisms Research Articles

A first-in-class selective inhibitor of EGFR and PI3K offers a single-molecule approach to targeting adaptive resistance.

Despite tremendous progress in precision oncology, adaptive resistance mechanisms limit the long-term effectiveness of molecularly targeted agents. Here we evaluated the pharmacological profile of MTX-531 that was computationally designed to selectively target two key resistance drivers, epidermal growth factor receptor and phosphatidylinositol 3-OH kinase (PI3K). MTX-531 exhibits low-nanomolar potency against both targets with a high degree of specificity predicted by cocrystal structural analyses. MTX-531 monotherapy uniformly resulted in tumor regressions of squamous head and neck patient-derived xenograft (PDX) models. The combination of MTX-531 with mitogen-activated protein kinase kinase or KRAS-G12C inhibitors led to durable regressions of BRAF-mutant or KRAS-mutant colorectal cancer PDX models, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to the hyperglycemia commonly seen with PI3K inhibitors. Here, we show that MTX-531 acts as a weak agonist of peroxisome proliferator-activated receptor-γ, an attribute that likely mitigates hyperglycemia induced by PI3K inhibition. This unique feature of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors.

Adaptive changes in the fatty acid composition of <i>Oxytropis scheludjakovae</i> and <i>Oxytropis strobilacea</i> under temperature fluctuations during the growth season in Central Yakutia

Although there is growing interest in understanding the mechanisms of plant adaptive resistance, there is still incomplete or missing information for many plant species. In this study, we conducted an analysis of the fatty acid composition in the vegetative and generative organs of two species belonging to the Oxytropis genus, namely O. scheludjakovae and O. strobilacea. Gas-liquid chromatography was used to analyze the qualitative and quantitative changes in fatty acids throughout the growing season. The plants were grown in the natural flora collection nursery at the Yakutsk Botanical Garden. The main fatty acids identified had carbon atoms ranging from 14 to 24. There were no significant differences in fatty acid composition between the endemic species O. scheludjakovae and the more widely distributed species O. strobilacea. The highest total fatty acid content (19.5 mg/g tissue) was observed in the leaves of O. scheludjakovae in June and in the leaves of O. strobilacea (17.0 mg/g tissue) in June and July. Palmitic acid was the primary saturated fatty acid in both species, while linolenic acid was the main unsaturated fatty acid. The adaptation of these plants to temperature changes was reflected in the variation of unsaturated fatty acid content. O. strobilacea showed higher values of fatty acid unsaturation coefficient throughout the growing season compared to O. scheludjakovae.

The evolving role of immune checkpoint inhibitors in cervical and endometrial cancer

The introduction of immune checkpoint inhibitors (ICIs) has revolutionized the treatment landscape for numerous tumor types, including cervical and endometrial cancers. Multiple ICIs against programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have demonstrated encouraging outcomes in controlled clinical studies for advanced cervical and endometrial cancers. For advanced cervical cancer, approved ICIs as second-line treatment include cemiplimab, nivolumab, and pembrolizumab as single agents. In the first-line treatment setting, options include pembrolizumab alone or in combination with bevacizumab, as well as atezolizumab combined with a backbone platinum-based chemotherapy plus bevacizumab. Additionally, for locally advanced cervical cancer, pembrolizumab is recommended alongside concurrent chemoradiotherapy. For endometrial cancer, pembrolizumab monotherapy, pembrolizumab in combination with lenvatinib, and dostarlimab are currently approved as second-line treatment options. Moreover, either dostarlimab or pembrolizumab can be added to first-line platinum-based chemotherapy for mismatch repair deficient malignancies. Although the inclusion of these agents in clinical practice has led to improved overall response rates and survival outcomes, many patients still lack benefits, possibly due to multiple intrinsic and adaptive resistance mechanisms to immunotherapy. This review aims to highlight the rationale for utilizing ICIs and their current role, while also delineating the proposed mechanisms of resistance to ICIs in cervical and endometrial cancer.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Abstract PO2-13-07: SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT

Abstract Objectives/Rationale: Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for disease recurrence and death. While multiple prognostic biomarkers associated with response to NAST have been identified, adaptive resistance mechanisms to NAST remain poorly understood. Identifying and characterizing adaptive resistance mechanisms can illuminate novel therapeutic approaches and improve outcomes for TNBC patients with RD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with RD) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. Comparative marker selection analysis was performed by computing the two-sided paired samples t-test for each gene followed by pre-ranked gene set enrichment analysis (GSEA) amongst 23,797 annotated gene sets. Gene sets with false-discovery rate (FDR) corrected P values < 0.001 were determined to be significant. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients with RD (N=27 from NeoSTOP and N=31 from NeoPACT). 165/23,797 gene sets were significantly enriched in post-treatment compared to pre-treatment samples. SHEN_SMARCA2_TARGETS_UP (M29) was among the top five significantly enriched gene set and was selected for further analysis based on the known role of the SWI/SNF complex in cell survival and the availability of clinically viable SMARCA2 degraders. Pre-treatment SMARCA4 expression was associated with pCR, with lower median expression noted in patients with RD compared to patients who achieved pCR (P=0.04). Pre-treatment SMARCA2 expression was not associated with pCR (P=0.48). On paired sample analysis there was pronounced downregulation of SMARCA4 in post-treatment compared to pre-treatment samples in all patients (mean Z=-1.46, P< 0.0001), in patients treated with chemotherapy (mean Z=-1.65, P< 0.0001), and in patients treated with chemoimmunotherapy (mean Z=-1.28, P< 0.0001). Although there was no difference in SMARCA2 expression between paired pre- and post-treatment samples (mean Z=0.03, P=0.83), there was enrichment of SMARCA2 target expression on GSEA analysis (NES=2.63, FDR q< 0.00001) indicating that SMARCA2 activity is compensating for therapy-induced SMARCA4 loss. Conclusions: We identified 165 gene sets that were significantly enriched in paired post-treatment compared to pre-treatment TNBC samples. Altered activity of the SMARCA2 subunit of the SWI/SNF chromatin remodeling complex was identified as one of the top hits. SMARCA2 and SMARCA4 are mutually exclusive subunits of SWI/SNF, and inactivation of both SMARCA2 and SMARCA4 is synthetic lethal. We observed an association of lower pre-treatment SMARCA4 expression with resistance to NAST and pronounced therapy-induced downregulation of SMARCA4 and induction of SMARCA2 target genes in post-treatment compared to pre-treatment samples, suggesting that SMARCA2 compensates for adaptive SMARCA4 loss. SMARCA2 degraders are in early-phase clinical trials and are known to induce synthetic lethality in SMARCA4-deficient cells. Our results identify adaptive loss of SMARCA4 and synthetic lethal targeting of compensatory SMARCA2 as an attractive therapeutic target in TNBC patients with RD after neoadjuvant chemotherapy or chemoimmunotherapy. Citation Format: Shane Stecklein, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-07.

Intraspecific variation in antibiotic resistance potential within E. coli.

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.

IGSF8 is an innate immune checkpoint and cancer immunotherapy target

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival invitro or invivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells invitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and Tcell signaling invivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.

Abstract LB293: O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance

Abstract Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs in melanoma. Deploying a model of adaptive cancer drug tolerance H3K4me3 ChIP-seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in vivo. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Gregory Gimenez, Elmar Zuegner, Christoph Magens, Michael Eccles, Mayumi Fujita, Helmut Schaider. O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB293.

Abstract 4666: HC-7366, a potent GCN2 kinase activator, augments osimertinib therapy to delay resistance in EGFR mutant NSCLC models

Abstract Despite the efficacy seen with EGFR tyrosine kinase inhibitors (TKIs) in the treatment of EGFR mutant NSCLC, the long-term prognosis remains unfavorable due to resistance. Osimertinib is used as the first- and second-line treatment for EGFR exon 19 deletion, L858R mutation, and acquired T790M mutation. Resistance is driven by both EGFR-dependent and independent events, such as gene amplification and upregulation of HIF1α. Metabolic events also drive resistance by increasing oxidative phosphorylation (OXPHOS) caused by osimertinib-induced glycolysis inhibition. HiberCell has developed HC-7366, a GCN2 activator currently being investigated in a Ph1 clinical trial in patients with solid tumors (NCT05121948). GCN2 activation by HC-7366 drives the integrated stress response (ISR) by inducing the expression of ATF4. This results in translation inhibition, cell cycle arrest, HIF1α suppression, and apoptosis in cancer cells. Our preclinical data suggest HC-7366 can reduce glycolysis, TCA cycle metabolites, and OXPHOS, thereby causing mitochondrial dysregulation. We hypothesized that combination of HC-7366 with osimertinib could reduce resistance by evading the adaptive resistance mechanisms. We treated the NCI-H1975 xenograft model with HC-7366, a clinically relevant dose of osimertinib, and the combination for 21 days and then followed for tumor regrowth. At day 21, the HC-7366 group showed efficacy with tumor growth inhibition (TGI) of 55%, while osimertinib resulted in tumor regression of 97% with 8/10 CRs and 2/10 PRs. The combination had tumor regression of 99% with 10/10 CRs. After treatment cessation for 74 days, tumors in the osimertinib group quickly regrew with 6/10 tumors progressing and 4/10 mice tumor-free. In contrast, the combination group had only 2/10 tumors regrow with 8/10 mice tumor-free. In an EGFR-dependent resistance model NCI-H1975 (EGFR C797S), combination achieved 63% TGI versus 31% with osimertinib. In an EGFR-independent resistant PDX with EGFR exon 19 deletion, combination benefit was seen at 70% TGI compared to 40% for osimertinib, while a PDX with EGFR L858R showed a trend for combination benefit. To understand the mechanism of action, we examined osimertinib-resistance pathways to determine how GCN2 activation could affect them. Combination with HC-7366 reduced the expression of short-lived proteins involved in osimertinib resistance, including HIF1α, MET, cyclin D1, and CDK4. Combination decreased TIM17A and TIM23, proteins essential for mitochondrial protein translocation, potentially causing mitochondrial dysfunction. We found a proliferation reduction by Ki67 and an increase in apoptotic proteins CHOP, PUMA, and cleaved caspase 3. Taken together, these data reveal that combination of HC-7366 and osimertinib, and potentially other EGFR TKIs, may be an effective treatment strategy at targeting adaptive resistance mechanisms. Citation Format: Crissy Dudgeon, Sho Fujisawa, Olivia Funk, Feven Tameire, Takashi O. Kangas, Weiyu Zhang, Eric S. Lightcap, David Surguladze, Nandita Bose. HC-7366, a potent GCN2 kinase activator, augments osimertinib therapy to delay resistance in EGFR mutant NSCLC 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 4666.

Abstract 6589: Discovery of potent and selective pan-TEAD autopalmitoylation inhibitors for the treatment of Hippo-pathway altered cancers

Abstract TEAD1-4 are DNA bound transcription factors regulated by the Hippo tumor suppressor pathway that bind the coactivators YAP/TAZ to activate oncogenic gene expression programs involved in cell survival, proliferation, and drug resistance. TEAD-dependent transcription is activated in many tumor types through a variety of mechanisms including LOF Hippo pathway mutations (e.g. NF2, LATS) and YAP/TAZ amplification, and also as an adaptive resistance mechanism, for example in response to EGFR TKI and KRAS inhibition. Here, we report the initial characterization of a novel pan-TEAD inhibitor, AZ4331, which was identified following a structure-guided drug design campaign. AZ4331 disrupts the post-translational palmitoylation of TEAD proteins through covalent binding to a conserved cysteine residue resulting in inhibition of TEAD-dependent transcriptional output. AZ4331 inhibited the proliferation of NF2m NCI-H226 cells (GI50: 92 nM) while having no effect on Hippo-wild type NCI-H2452 (GI50: undetermined). Cell-based metabolic labeling studies demonstrated the ability of AZ4331 to inhibit palmitoylation of all four TEAD paralogs and led to disruption of the YAP/TEAD complex assessed by immunoprecipitation resulting in reduced TEAD transcriptional output as assessed by qPCR. Furthermore, in vivo profiling showed AZ4331 achieved sufficient exposures (plasma free Cmax: 2.7 µM) and robust target engagement as measured through reduction of multiple canonical TEAD target genes (CTGF [IC50: 0.127 µM], ANKRD1, AMOTL2 and CYR61) in Hippo-altered mesothelioma xenograft tumors. This target engagement in turn drove inhibition of tumor proliferation in xenograft models representing common Hippo-alterations. AZ4331 treatment of NF2m NCI-H226 and LATS1/2 loss MSTO-211H mesothelioma xenografts drove 50% and 93% regression of tumors, respectively. While treatment of head and neck squamous cell carcinoma FAT1m FaDu and YAP1amp Detroit562 xenografts lead to 58% tumor growth inhibition and 35% tumor regression, respectively. These findings identify AZ4331 as a potent inhibitor of the TEAD transcription factor family and further validate the inhibition of TEAD in Hippo-altered cancers as a potential therapeutic strategy. Citation Format: Jacob A. Gordon, Jolanta Dubauskaite, Michelle DuPont, Nin Guan, Geoff Holdgate, Scott Mlynarski, Neil Umbreit, Danielle Sanchez, Abhishek Srivastava, Janek Suski, Nancy Su, Ryan Richards, Sabina Cosulich, Corinne Reimer, James Brownell. Discovery of potent and selective pan-TEAD autopalmitoylation inhibitors for the treatment of Hippo-pathway altered 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 6589.

Abstract 7194: Targeting AXL and MCL-1 to eradicate lazertinib tolerance in EGFR-mutated NSCLC cells

Abstract Lazertinib, a novel third-generation EGFR-tyrosine kinase inhibitor, has shown marked efficacy in patients with EGFR-mutant lung cancer. However, after various periods of therapeutic intervention, most patients eventually acquire resistance to lazertinib. A potential therapeutic approach to address this clinical issue is to overcome the initial drug resistance mechanism. To determine the underlying adaptive lazertinib resistance mechanisms, we evaluated signal transduction, cell apoptosis, and drug sensitivity in mutant EGFR-expressing non-small cell lung cancer (NSCLC) cells treated with lazertinib. The siRNA screening assay demonstrated that AXL knockdown significantly inhibited cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors eventually led to the proliferation of residual cells and increased MCL-1 expression, which was mediated by the nuclear translocation of the transcription factor YAP. Cell-based assays showed that triple therapy with an MCL-1 or YAP inhibitor, in combination with EGFR-TKI lazertinib, an AXL inhibitor, significantly reduced cell viability and increased apoptosis. These results demonstrate that AXL and YAP/MCL-1 signals contribute to the adaptive lazertinib resistance in EGFR-mutant NSCLC cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be highly effective in eliminating lazertinib-resistant cells. Citation Format: Tadaaki Yamada, Yohei Matsui, Koichi Takayama. Targeting AXL and MCL-1 to eradicate lazertinib tolerance in EGFR-mutated NSCLC cells [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 7194.

Abstract 6909: KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer models

Abstract Introduction: One quarter of lung adenocarcinomas (LUAD) harbor KRAS-mutations, with KRASG12C accounting for the majority (40%). GDP-KRASG12C inhibitors (G12Ci) have been developed for this patient cohort, and two of these are approved for clinical use in the US. While response rates to G12Ci tested in clinical trials have been between 35-50%, studies have identified mechanisms of adaptive resistance to these novel agents, limiting their use as monotherapies. However, primary (intrinsic) resistance to these compounds has not been explored extensively. We leveraged our patient-derived xenograft (PDX) and xenograft-derived organoid (XDO) development programs to study primary resistance to the novel G12Ci, AZD4625. Methods: Twelve KRASG12C PDX models were established which recapitulated patient tumor histology, and genomic, transcriptomic, and methylome profiles. From these 12 PDX, six long-term (passage >10) XDO were generated. PDX were treated with AZD4625 chronically for four weeks as well as in an acute dosing pharmacodynamic study, where tumors were harvested for further analysis. Results: The AZD4625 drug screen in our PDX models reproduced the G12Ci response rate observed in clinical trials. Four of twelve tumor models reduced in size on treatment, while the remainder were considered resistant as these tumors increased in size on treatment. Sensitive tumors became necrotic while those resistant remained proliferative. XDO models recapitulated their originating PDX genomic alterations, histology, as well as responses to AZD4625. During the acute pharmacodynamic screen in our PDX models, we observed a decrease in pERK1/2 and pS6 protein expression in sensitive but not resistant models, despite acute DUSP6 gene expression decreases in every model. Global proteomic analysis highlighted differences between each model. Conclusion: PDX and XDO are useful models to study resistance to this novel class of inhibitor, with the KRASG12C NSCLC PDX models exhibiting a treatment response rate that is similar to those observed in clinical trials. Global proteomics suggests uniqueness of each model in our cohort, hinting that a universal combination of specific targeted agents to treat KRASG12C LUAD tumors may be insufficient. Citation Format: Joshua C. Rosen, Nhu-An Pham, Quan Li, Pinjiang Cao, Katrina Hueniken, Takamasa Koga, Nikolina Radulovich, Alex Koers, Michael Niedbala, Sarah Ross, Adrian Sacher, Ming-Sound Tsao. KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer 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 6909.

Abstract 7534: Tobemstomig, a novel bispecific checkpoint inhibitory antibody to preferentially block PD-1 and LAG-3 on CD8 TILs over Tregs

Abstract Checkpoint inhibitors targeting PD-1 have shown unprecedented clinical efficacy in several cancer indications and therefore have, revolutionized the standard of care. However, despite this advancement, only ~20-30% of the patients derive durable benefit from such a treatment. One of the suggested reasons for this limited success is the expression/activation of compensatory inhibitory pathways such as LAG-3 on tumor-reactive T cells. Therefore, it is envisioned that simultaneous antagonism of PD-1 and LAG-3 receptors would overcome this adaptive resistance mechanism and allow a more profound reinvigoration of tumor-reactive T cells. LAG-3 is constitutively expressed by regulatory T cells (on Tregs) and it has been reported that its blockade increases Treg suppressive function which could potentially off-sets the benefit of blocking LAG-3 on tumor-reactive CD8 T cells.We therefore developed a 1+1 PD1-LAG3 bispecific antibody, Tobemstomig, with a unique PD-1 binder recognizing a glycoepitope and more than 20 fold higher affinity than the LAG-3 binder which recognizes a novel and differentiated binding epitope (E3). This difference in affinities allows for an avidity driven selectivity gain to PD-1 and LAG-3 double positive T cells over and the following advantages over monospecific and other bispecific aPD-1/-L1 and aLAG-3 antibodies:1.improved targeting to tumor-reactive T cells rather than Tregs due to the selectivity gain and different expression patterns of PD-1 and LAG-3 on these two T cell types, 2.reduced internalization, 3.resistance to drug-shaving by macrophages due to Fc -silencing via P329G LALA,4.increased in vitro T cell effector functions even in the presence of Tregs, 5.in-vivo targeting and expansion of CD8 TILs, including stem-like T cells, and increase in T cell effector functions and6.superior in vivo tumor control/eradication compared to combination of monospecific anti-PD1 and anti-LAG3 antibodies,in several mouse models including Panc02 in syngeneic huPD-1xhuLAG3 dtg mice and BXPC3, WSU, OCI-Ly18 and BC004 in humanized mice Tobemstomig has completed dose escalation and is currently tested in a composite proof of concept Ph2 trial in several solid tumor indications. Citation Format: Laura Codarri-Deak, Patrick Aaron Weber, Valeria Nicolini, Christian Klein, Pablo Umana, Stefan Seeber, Mario Perro, Stephane Leclair. Tobemstomig, a novel bispecific checkpoint inhibitory antibody to preferentially block PD-1 and LAG-3 on CD8 TILs over Tregs [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 7534.

Abstract 1230: MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance

Abstract Adaptive resistance mechanisms compromise the long-term effectiveness of kinase-targeted agents dictating the need for strategic combination approaches. MTX-531 was computationally designed to selectively target both PI3K and wild type EGFR. MTX-531 exhibits low nanomolar potency against the PI3K isoform family and EGFR with a high degree of specificity predicted by co-crystal structural analyses. Full kinome screening confirmed that MTX-531 is exquisitely selective for its intended targets. We evaluated the pharmacological profile of MTX-531, whereupon single agent treatment with this lead molecule elicited a high incidence of durable tumor regressions in a broad panel of PIK3CA mutant CDX and PDX squamous head and neck (HNSCC) models. The combination of MTX-531 with RAS pathway inhibitors, including agents directed against KRAS G12C, led to durable regressions of KRAS mutant colorectal and pancreatic xenografts, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to hyperglycemia and hyperinsulinemia commonly seen with predecessor pan PI3K inhibitors. MTX-531 acts as an agonist of PPARγ, a structural feature of the molecule that is thought to mitigate hyperglycemia induced by PI3K inhibition. This unique attribute of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors. To the best of our knowledge, MTX-531 is the first reported pan-PI3K inhibitor that does not lead to hyperglycemia. The pharmaceutical profile of MTX-531 includes favorable cross-species oral bioavailability, microsomal stability, and a facile 3 step chemical synthesis. MTX-531, which has the unique capability of concurrently and selectively inhibiting PI3K and EGFR, illustrates the power of rational computational drug design to target multiple adaptive resistance mechanisms in a single molecule. The versatility of MTX-531 in both the single agent and combination settings offers a breadth of development strategies that continue to evolve as new combination candidates become available. MTX-531 is currently undergoing advanced preclinical development for anticipated first in human clinical trials in late 2024. Citation Format: Christopher E. Whitehead, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Rachel A. Mumby, June Chung, Jinju Li, Nathaniel Osher, Oluwadara Coker, Michelle Norris, Scott Kopetz, Veera Baladandayuthapani, Melinda Hollingshead, Sharad Verma, Judith S. Sebolt-Leopold. MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance [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 1230.

Abstract 1969: Off-target autophagy inhibition by SHP2 allosteric inhibitors and therapeutic implications for aberrant RAS-driven cancers

Abstract Aberrant activation of RAS-MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in a SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS-MAPK pathway blockage. Finally, we exemplified a new therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS driven and drug resistant malignancies such as pancreatic and colorectal cancers. Citation Format: Yiming Miao, Yunpeng Bai, Jinmin Miao, Allison A. Murray, Jianping Lin, Jiajun Dong, Zihan Qu, Ruo-Yu Zhang, Quyen Nguyen, Shaomeng Wang, Zhong-Yin Zhang. Off-target autophagy inhibition by SHP2 allosteric inhibitors and therapeutic implications for aberrant RAS-driven 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 1969.

Abstract 4653: Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in NSCLC

Abstract Genetic profiling and the development of targeted therapy has improved survival of many cancer patients, but resistance to targeted therapy remains a major barrier to long-term patient survival. To develop more pro-active strategies to improve clinical outcomes, our research focuses on drug-tolerant persister cells that constitute a reservoir of cancer cells that survive and adapt during the initial stage of therapy. These cells typically display therapy resistance in a reversible manner and set the stage for the subsequent emergence of cells with full acquired resistance. We utilized quantitative proteomics to characterize persister cells generated from EGFRmut, KRASmut, and ALKmut non-small cell lung cancer (NSCLC) cell lines after treatment with osimertinib, sotorasib, and lorlatinib, respectively. Integrating proteomics data with network analysis revealed a decrease in the signaling network associated with cell cycle regulation and DNA replication. Meanwhile, persister cells exhibited an enrichment in proteins linked to lysosomes, amino acids, and fatty acid metabolism. The data revealed a consistent metabolic shift in persister cells, with an increased dependence on oxidative phosphorylation (OXPHOS) for ATP generation. Small-scale drug screening was performed based on findings from proteomics and confirmed that persister cells are vulnerable to inhibitors of OXPHOS mitochondrial complexes. Combination treatment of OXPHOS inhibitors and each targeted therapy delayed the emergence of resistance. One complex V inhibitor, Gboxin, selectively inhibited the growth of persister cells. The therapeutic potential of complex V was further validated by CRISPR-based genetic knockdown studies. The metabolic transition in drug tolerant persister cells may serve as a critical adaptive resistance mechanism to provide a survival advantage under targeted therapy-induced stress. Understanding this metabolic shift may lead to new treatments that aim to prevent or delay the onset of acquired resistance in NSCLC. Citation Format: Donghwa Kim, Dmitry Kuchenov, Aubhishek Zaman, Neal Bennett, Emilio Ramos, Ken Nakamura, Arun P. Wiita, Sourav Bandyopadhyay, Trever Bivona. Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in 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 4653.

Abstract A065: High throughput drug screening to decipher the mechanisms of trametinib adaptive resistance in low-grade serous ovarian cancer cell lines

Abstract Low Grade Serous Ovarian Cancer (LGSOC) is a rare disease but accounts for a significant percentage of epithelial ovarian cancers in young women. Targeted therapy with trametinib, a MEK inhibitor (MEKi), has emerged as a new treatment option for women with recurrent LGSOC from a recent clinical trial. Initial tumor shrinkage was observed in approximately 26% patients treated with trametinib but most patient eventually died of the disease because the tumor became trametinib adaptive resistance. Thus, further investigation to overcome trametinib resistance is necessary to improve the survival of patients with LGSOC. To understand how LGSOC patients develop adaptive resistance to trametinib, we generated several trametinib adaptive resistant LGSOC cell lines. By comparing the expression of over 400 signaling proteins with reverse phase protein array (RPPA) between an adaptive resistant cell (HOC-7R) to the parental cells (HOC-7), we discovered several proteins that were up-regulated in the adaptive resistant cell lines such as plasminogen activator inhibitor-1 (PAI-1 or SERPINE1), p-Rb, p-ERK1/2, MEK1, p-S6, p-Yb1, p-YAP, p-Chk1, and histone H3. By over-expressing SERPINE1 in cell line HOC-7, we were able to show that HOC-7-SERPINE1 (a stable clone of HOC-7 overexpressing SERPINE1) became more resistant to trametinib. By high-throughput drug screening with 555 drugs from libraries with targeted molecules, approved drugs, investigational agents, and bioactive molecules, HOC-7-SERPINE1 cells were confirmed to have increased resistance to other MEK inhibitors in comparison to the parental cell HOC-7. The fold increase in IC50 for MEKi for HOC-7-SERPINE1 were 3.46 (18.65 nM/5.39 nM), 3.74 (28.74 nM/7.68 nM), 2.65 (52.2 nM/19.68 nM) and 1.66 (22.79 nM/13.73 nM) for trametinib, binimetinib, selumetinib and cobimetinib respectively. In addition, overexpression of SERPINE1 in HOC-7 cells also enhanced their resistance to an SRC inhibitor (saracatinib; about 1.7 folds increase in IC50), histone deacetylases inhibitor (belinostat; about 1.2 folds increase in IC50) and Bcl-2 inhibitor (obatoclax; about 2.38 folds increase in IC50). On the other hand, HOC-7-SERPINE1 became more sensitive to farnesyltransferase inhibitor (lonafarnib; about 36.8 folds decrease in IC50), PARP (Poly ADP Ribose Polymerase) inhibitor (talazoparib; about 2.69 folds decrease in IC50). These results suggest that HOC-7-SERPINE1 cells become less dependent on SRC, histone deacetylases, and Bcl-2 than the parental HOC-7 cells but become more dependent on signaling through farnesyltransferase and PARP. The combination of MEKi and PARPi (PARP inhibitor) has previously been shown to be a promising combination and is in clinical trial. It is possible that combinations of trametinib and lonafarnib treatment could overcome trametinib adaptive resistance. In conclusion, high-throughput drug screening allows us to discover potential mechanisms of trametinib adaptive resistance and potential drugs for treatment of LGSOC patients who have developed trametinib resistance. Citation Format: Kwong-Kwok Wong, Chun Wai Ng, Yvonne Tsang, David M. Gershenson. High throughput drug screening to decipher the mechanisms of trametinib adaptive resistance in low-grade serous ovarian cancer cell lines [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 A065.

Adaptive Darwinian off-target resistance mechanisms to selective RET inhibition in RET driven cancer

Patients treated with RET protein tyrosine kinase inhibitors (TKIs) selpercatinib or pralsetinib develop RET TKI resistance by secondary RET mutations or alterative oncogenes, of which alterative oncogenes pose a greater challenge for disease management because of multiple potential mechanisms and the unclear tolerability of drug combinations. A patient with metastatic medullary thyroid carcinoma (MTC) harboring a RET activation loop D898_E901del mutation was treated with selpercatinib. Molecular alterations were monitored with tissue biopsies and cfDNA during the treatment. The selpercatinib-responsive MTC progressed with an acquired ETV6::NTRK3 fusion, which was controlled by selpercatinib plus the NTRK inhibitor larotrectinib. Subsequently, tumor progressed with an acquired EML4::ALK fusion. Combination of selpercatinib with the dual NTRK/ALK inhibitor entrectinib reduced the tumor burden, which was followed by appearance of NTRK3 solvent-front G623R mutation. Preclinical experiments validated selpercatinib plus larotrectinib or entrectinib inhibited RET/NTRK3 dependent cells, whereas selpercatinib plus entrectinib was necessary to inhibit cells with RET/NTRK3/ALK triple alterations or a mixture of cell population carrying these genetic alterations. Thus, RET-altered MTC adapted to selpercatinib and larotrectinib with acquisition of ETV6::NTRK3 and EML4::ALK oncogenes can be managed by combination of selpercatinib and entrectinib providing proof-of-concept of urgency of incorporating molecular profiling in real-time and personalized N-of-1 care transcending one-size-fits-all approach.

Discovery of bioactive natural products of microbial origin as inhibitors of the PD-1/PD-L1 protein-protein interaction

The PD-1/PD-L1 protein-protein interaction (PPI) controls an adaptive immune resistance mechanism exerted by tumor cells to evade immune responses. The large-molecule nature of current commercial monoclonal antibodies against this PPI hampers their effectiveness by limiting tumor penetration and inducing severe immune-related side effects. Synthetic small-molecule inhibitors may overcome such limitations and have demonstrated promising clinical translation, but their design is challenging. Microbial natural products (NPs) are a source of small molecules with vast chemical diversity that have proved anti-tumoral activities, but which immunotherapeutic properties as PD-1/PD-L1 inhibitors had remained uncharacterized so far. Here, we have developed the first cell-based PD-1/PD-L1 blockade reporter assay to screen NPs libraries. In this study, 6000 microbial extracts of maximum biosynthetic diversity were screened. A secondary metabolite called alpha-cyclopiazonic acid (α-CPA) of a bioactive fungal extract was confirmed as a new PD-1/PD-L1 inhibitor with low micromolar range in the cellular assay and in an additional cell-free competitive assay. Thermal denaturation experiments with PD-1 confirmed that the mechanism of inhibition is based on its stabilization upon binding to α-CPA. The identification of α-CPA as a novel PD-1 stabilizer proves the unprecedented resolution of this methodology at capturing specific PD-1/PD-L1 PPI inhibitors from chemically diverse NP libraries.