Mechanisms Of Acquired Resistance Research Articles

Osimertinib and palbociclib in an EGFR-mutated NSCLC with primary CDK4 amplification after progression under osimertinib

Precision cancer medicine has changed the treatment paradigm of patients with non-small cell lung cancer (NSCLC) with specific molecular aberrations. A major challenge is management of the resistance that tumor cells eventually develop against targeted therapies, either through primary or acquired resistance mechanisms. We report a 61 year-old male patient with metastatic NSCLC harboring an EGFR exon 19 deletion, a PIK3CA mutation, and CDK4 amplification. After an initial partial response to osimertinib as mono-therapy (third-generation EGFR tyrosine kinase inhibitor), the patient had progression of disease after 4 months of treatment and was referred for combined osimertinib and palbociclib (CDK4/6 inhibitor) treatment. Though complicated by transient pneumonitis, the patient has an ongoing partial response for > 10 months and has experienced clinical improvement on this treatment regimen. As amplification of CDK4 occurs in ~ 10% of treatment-naïve patients with EGFR-mutated NSCLC, the successful treatment of our patient with osimertinib and palbociclib may be highly relevant for future patients with NSCLC.

Quantitative systems pharmacology modeling of tumor heterogeneity in response to BH3-mimetics using virtual tumors calibrated with cell viability assays.

Both primary and acquired resistance mechanisms that involve intra-tumoral cell heterogeneity limit the use of BH3-mimetics to trigger tumor cell apoptosis. This article proposes a new quantitative systems pharmacology (QSP)-based methodology in which cell viability assays are used to calibrate virtual tumors (VTs) made of virtual cells whose fate is determined by simulations from an apoptosis QSP model. VTs representing SU-DHL-4 and KARPAS-422 cell lines were calibrated using invitro data involving venetoclax (anti-BCL2), A-1155463 (anti-BCLXL), and/or A-1210477 (anti-MCL1). The calibrated VTs provide insights into the combination of several BH3-mimetics, such as the distinction between cells eliminated by at least one of the drugs (monotherapies) from the cells eliminated by a pharmacological combination only. Calibrated VTs can also be used as initial conditions in an agent-based model (ABM) framework, and a minimal ABM was developed to bridge invitro SU-DHL-4 cell viability results to tumor growth inhibition experiments in mice.

KRAS G12C inhibitor combination therapies: current evidence and challenge.

Although KRAS G12C inhibitors have proven that KRAS is a "druggable" target of cancer, KRAS G12C inhibitor monotherapies have demonstrated limited clinical efficacy due to primary and acquired resistance mechanisms. Multiple combinations of KRAS G12C inhibitors with other targeted therapies, such as RTK, SHP2, and MEK inhibitors, have been investigated in clinical trials to overcome the resistance. They have demonstrated promising efficacy especially by combining KRAS G12C and EGFR inhibitors for KRAS G12C-mutated colorectal cancer. Many clinical trials of combinations of KRAS G12C inhibitors with other targeted therapies, such as SOS1, ERK, CDK4/6, and wild-type RAS, are ongoing. Furthermore, preclinical data have suggested additional promising KRAS G12C combinations with YAP/TAZ-TEAD inhibitors, FAK inhibitors, and farnesyltransferase inhibitors. The combinations of KRAS G12C inhibitors with immunotherapies and chemotherapies have also been investigated, and the preliminary results were reported. More recently, KRAS-targeted therapies not limited to KRAS G12C are being developed, potentially broadening the treatment landscape of KRAS-mutated cancers. Rationally combining KRAS inhibitors with other therapeutics is likely to play a significant role in future treatment for KRAS-mutated solid tumors.

Abstract PO2-16-05: ESR1 mutations (ESR1mut) in HR(+)HER2(-)patients with metastatic breast cancer (MBC): prevalence along treatment course and predictive value for endocrine therapy (ET) resistance in real-world practice

Abstract Background: ET combined with CDK4/6 inhibitors (CDKi) is the standard of care for HR(+)HER2(-) patients with MBC. ESR1mut is an acquired resistance mechanism to ET, especially aromatase inhibitors (AI). The prevalence and behavior of ESR1mut in patients receiving the selective estrogen receptor degrader (SERD) fulvestrant remains in question. This study aimed to characterize the prevalence of ESR1mut at start of successive lines of therapy and to evaluate clinical outcomes of ET by ESR1 status in 1st line therapy in real-world practice. Methods: This study included patients with HR(+)HER2(-) MBC who underwent genomic testing using Foundation Medicine tissue or liquid comprehensive genomic profiling (CGP) assays, with specimens collected within 60 days of therapy initiation. Patient clinical data was obtained by the nationwide (US-based) de-identified Flatiron Health and Foundation Medicine real-world clinicogenomic breast database (FH-FMI CGDB), originated from ~280 US cancer clinics (~800 sites of care) between 01/2011 and 04/2023. ESR1mut prevalence was calculated in tissue and liquid samples collected in the 1st, 2nd, and 3rd lines of therapy. Real-world progression free-survival (rwPFS), time to treatment discontinuation (rwTTD), and overall survival (rwOS) were compared between patients who received AI + CDKi and between patients receiving fulvestrant + CDKi 1st line therapy [ESR1mut vs. ESR1 wild-type (wt) by tissue CGP] by Cox models. Multivariable analyses adjusted for adjuvant therapy, age, ECOG, menopausal status, histology, stage at diagnosis, metastatic site, and specific ESR1mut (Y537S, D538G, E380W, or others) were performed. Results: The prevalence of ESR1mut in tissue samples collected in 1st, 2nd, and 3rd lines of therapy was 7.9% (n=159/2004), 26.4% (n=66/250), and 34.0% (n=68/200), respectively. The prevalence of ESR1mut in liquid samples collected in the 1st, 2nd, and 3rd lines of therapy was 11.6% (n=30/259) 34.4% (n=63/183), and 35.9% (n=42/117), respectively. Among patients receiving AI + CDKi 1st line therapy (n=485), those with ESR1mut vs. ESR1wt had less favorable rwPFS [median 6.1 vs. 20.8 months, hazard ratio (HR) 2.36, 95% CI 1.35-4.14, p=0.003]. rwTTD and rwOS assessments were consistent with the magnitude of effect of rwPFS. Among patients receiving fulvestrant + CDKi 1st line therapy (n=302), those with ESR1mut vs. ESR1wt had less favorable rwPFS (median 10.5 vs. 14.5 months, HR 1.5, 95% CI 1.05-2.15, p=0.025), but no difference was observed for rwTTD and rwOS (p >0.05). Notably, the multivariable analyses found the independent association of ESR1mut and less favorable rwPFS (HR 2.18, 95% CI 1.15-4.1, p=0.018), rwTTD (HR 3.34, 95% CI, 1.84-6.1, p< 0.001), and rwOS (HR 2.46, 95% CI 1.04-5.9, p=0.041) in patients receiving AI + CDKi. The multivariable analyses confirmed no association between ESR1mut and outcomes in patients receiving fulvestrant + CDKi (p >0.05). No substantial differential associations were observed between specific ESR1mut and clinical outcomes in the multivariable analyses. Conclusion: In our dataset, ESR1mut is prevalent in about 8-11% of baseline tissue and liquid samples of patients with HR(+)HER2(-) MBC. Baseline ESR1mut is associated with less favorable outcomes in patients receiving AI + CDKi 1st line therapy, but not in patients receiving fulvestrant + CDKi. Specific ESR1mut do not seem to be associated with different outcomes to ET. ESR1mut prevalence increased during treatment course. Further investigation is warranted to determine optimal endocrine partner with CDKi based upon ESR1 status, particularly with development of oral SERDs. Citation Format: Manali Bhave, Julia Quintanilha, Ryon Graf, Takara Scott, Hanna Tukachinsky, Gerald Li, Alexa Schrock, Geoffrey Oxnard, Mia Levy, Kevin Kalinsky. ESR1 mutations (ESR1mut) in HR(+)HER2(-)patients with metastatic breast cancer (MBC): prevalence along treatment course and predictive value for endocrine therapy (ET) resistance in real-world practice [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 PO2-16-05.

Addressing the Challenges of PD-1 Targeted Immunotherapy in Cancer Treatment

Background: Immune checkpoint inhibitors targeting the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis have revolutionized cancer treatment, demonstrating remarkable clinical efficacy across multiple malignancies. However, their broader applicability and therapeutic potential remain constrained by several challenges. Objective: This review aims to provide a comprehensive overview of the current challenges associated with PD-1 targeted immunotherapy, encompassing resistance mechanisms, patient selection, toxicity management, and the development of novel combination strategies. Methods: A comprehensive literature search was conducted across PubMed, Embase, and Web of Science databases to identify and critically analyze studies investigating the challenges and limitations of PD-1 targeted immunotherapy in cancer treatment. Results: The primary challenges identified include: (1) Intrinsic and acquired resistance mechanisms, such as alterations in antigen presentation, tumor microenvironment factors, and immune cell dysfunction; (2) Lack of reliable predictive biomarkers for patient selection and response monitoring; (3) Immune-related adverse events and their effective management; (4) Optimization of combination regimens with other immunotherapies, targeted therapies, or conventional treatments; (5) Financial and accessibility barriers limiting the broader implementation of immunotherapy. Conclusion: PD-1 targeted immunotherapy has transformed cancer treatment, addressing the current challenges is imperative to maximize its therapeutic potential and broaden its applicability. Ongoing research efforts focused on elucidating resistance mechanisms, developing predictive biomarkers, managing toxicities, and exploring novel combination strategies hold promise for overcoming these limitations. Interdisciplinary collaborations and continued investment in immunotherapy research are essential to ensure that the benefits of these groundbreaking therapies are accessible to a wider patient population.

Abstract 6056: Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-driven cancers

Abstract Monotherapies which target nodes within the RTK-RAS-MAPK pathway often display early potency in the clinic, but their preliminary response is limited as tumors begin to regrow due to adaptive and acquired resistance mechanisms. Disrupting proteins that mitigate either type of resistance mechanism in combination with oncogene-targeted agents could help extend and enhance the clinical potential of these drugs. Son of Sevenless 1 (SOS1), a RAS guanine nucleotide exchange factor, is a highly attractable protein to target given its role in transducing inputs from upstream receptor tyrosine kinases (RTKs) to downstream RAS proteins to regulate signaling pathways involved in cell proliferation and survival. Additionally, SOS1 is subject to MAPK-mediated negative feedback inhibition and drugs targeting upstream and downstream nodes relieves SOS1 from this inhibition resulting in pathway reactivation. To address this need, we have developed orally bioavailable SOS1 bifunctional degraders for cancers driven by KRAS and RTKs. Our top two lead molecules demonstrate CRBN- and proteasomal-mediated SOS1 degradation with DC50s < 10nM and maximum degradation > 90%. Consistent with this notion, SOS1 degradation inhibited downstream signaling marker phopshoERK with IC50s < 5nM and were 26- or 53-fold more potent when compared to the clinical SOS1 inhibitor. Notably, the SOS1 degraders exhibited weaker SOS1 binding compared to other SOS1 inhibitors highlighting that the potency is heavily driven by the catalytic nature of degradation rather than occupancy-driven events. Both SOS1 degraders displayed CRBN- and SOS1-dependent antiproliferative activity in KRAS- and EGFR- mutant, solid tumor cell lines as well as in BCR-ABL driven, CML cells with IC50 values ranging from 0.5-10nM. Consistent with our in vitro data, oral administration of our SOS1 degraders in mice harboring H358 (KRAS G12C) and H441 (KRAS G12V) xenografts resulted in SOS1 degradation, phosphoERK reduction and significant tumor growth inhibition. While SOS1 degrader monotherapy demonstrated in vitro and in vivo potency, combination of mutant or allele specific EGFR and KRAS inhibitors and ABL inhibitors with our proof-of-concept degraders prevented pathway reactivation associated with these inhibitors and lead to a more durable response. These combinations also yielded synergistic effects in in vitro proliferation assays as well as enhanced tumor growth inhibition in KRAS-mutant xenograft models. Together, our SOS1 degraders alone and in combination with other targeted agents demonstrated anti-tumor activity in a variety of KRAS- and RTK-driven cancer cell lines and xenograft models. These results support the potential of orally bioavailable SOS1 degraders to help mitigate pathway reactivation for enhanced efficacy and prolonged response duration. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Elena Martinez, Qiao Liu, Arvind Shakya, Akinori Okano, Venkat Mali, Shenlin Huang, Aparajita Chourasia, Leah Fung. Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-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 6056.

Abstract 1989: Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment

Abstract Introduction: Non-small cell lung cancers (NSCLC) harboring EGFR mutations are commonly treated with EGFR tyrosine kinase inhibitors (TKIs), but the emergence of drug resistance often leads to treatment failure. While various acquired resistance mechanisms have been identified, they are not always actionable. Furthermore, since drug resistance is typically studied after cancer relapse, little is known about the cellular or microenvironmental taking place during the formation of resistance at the stage of minimal residual disease (MRD). Therefore, it is necessary to understand the mechanisms leading to resistance during MRD and develop new ways to prevent drug resistance in EGFR-mutant cancers. Methods: In this study, we discovered pre-existing intratumor heterogeneity in EGFR expression within EGFR-mutant NSCLCs and found that patient tumors contain clones with low mutant EGFR expression. To study the effect of this intratumor heterogeneity on drug response, we isolated low and high expressing cells and conducted longitudinal drug challenge assays in ex vivo and in vivo models. To dissect how the observed heterogeneity affects drug resistance and the tumor microenvironment during MRD, we employed patient-derived tumor cells and used novel tumor-on-a-chip co-culture approaches. Results: Our findings reveal that EGFR-low cells are more tolerant to drug treatment and can survive the initial TKI insult, making them more likely to contribute to drug resistance in EGFR-mutant NSCLCs. Additionally, the surviving EGFR-low cells were able to modify the tumor microenvironment leading to fibroblast accumulation and immune suppression, contributing to the drug-tolerant tumor microenvironment. Importantly, we found that pharmacological induction of EGFR using epigenetic inhibitors was able to sensitize the drug tolerant cells to EGFR inhibition, suggesting a potential strategy to overcome TKI resistance in NSCLC patients. Conclusions: In conclusion, our study sheds light on the critical role of intratumoral heterogeneity in treatment resistance and offers valuable insights into potential strategies to overcome this challenge. Our findings suggest that combination therapies that can alter the cellular phenotype during MRD have the potential to prevent intrinsic drug resistance. This underscores the importance of gaining a better understanding of the mechanisms underlying drug-tolerant microenvironment and developing more effective treatment strategies for patients with EGFR-mutant NSCLCs. Citation Format: Heidi M. Haikala, Bassel Alsaed, Jieun Son, Linh Lin, Prafulla Gokhale, Pasi Jänne. Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment [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 1989.

Abstract 2697: Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system

Abstract Cancer immunotherapy has shown noticeable clinical efficacy in many cancer types, while clinical benefits are only observed in 20-40% of treated patients. Therefore, combination cancer immunotherapy targeting multiple factors that limit antitumor immunity has been tested in the clinic. Accumulating evidence reveals that cancers that harbor limited immune cell infiltration (so-called immunologically cold tumors) respond poorly to current immunotherapy. Preclinical studies using animal models have shown that activation of innate immunity by TLR agonists or STING agonists can successfully remodel the TME to effectively induce antitumor immunity in those immunologically cold tumors. However, unexpectedly, all TLR agonists and STING agonists tested have not shown clinical efficacy in combination with immune checkpoint blockade therapy, including PD-1/PD-L1 blockade therapy. Hence, we addressed the mechanism(s) why stimulators of innate immunity failed to elicit effective antitumor immunity in cancer patients. We then employed OK-432, which reportedly stimulates TLR-2, TLR-4, and/or TLR-9, as OK-432 is widely used in Japan in the clinic. In vitro OK-432 treatment accelerated the maturation DCs, and efficiently induced CD8+ T cells in humans. However, the combination treatment with anti-PD-1 mAb and OK-432 failed to augment antitumor immunity in animal models using anti-PD-1 mAb monotherapy-resistant tumors; the data mirrors the failure of clinical trials of combination therapy with immune checkpoint blockade and TLR agonists or STING agonists in humans. Therefore, using the animal models, we explored the acquired resistance mechanism developed after combination treatment. While OK-432 treatment effectively stimulated DCs, innate immune suppressive cells, such as MDSCs, were concurrently accumulated in the TME. The accumulation of PMN-MDSCs after OK-432 treatment was induced in a CXCL1-CXCR2-dependent fashion. Accordingly, triple combination treatment with a PMN-MDSC inhibitor along with OK-432 and anti-PD-1 mAb attenuated the accumulation of PMN-MDSCs in the TME and improved the antitumor effect of the combination treatment. Taken together, we propose that successful combination treatment stimulating innate immunity against immunogenically cold cancers requires controlling the unwanted co-activation of innate immune suppressive cells. This study clearly demonstrates the mechanism of acquired resistance after administration of reagents that activate innate immunity, such as TLR agonists or STING agonists, and provides the basis for future successful combination therapy. Citation Format: Hitomi Nishinakamura, Sayoko Shinya, Takuma Irie, Shugo Sakihama, Tadaaki Ioroi, Takeo Naito, Keisuke Watanabe, Daisuke Sugiyama, Motohiro Tamiya, Tatsuya Yoshida, Tetsunari Hase, Takao Yoshida, Kennosuke Karube, Yuka Maeda, Shohei Koyama, Hiroyoshi Nishikawa. Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system [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 2697.

Abstract 6584: Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer

Abstract While checkpoint inhibitor treatment has extended the survival of many cancer patients, most initial responders will later develop disease progression through a variety of poorly understood acquired resistance mechanisms (CPI-AR). One such recently described mechanism involves the transcriptional hyperactivation of genes associated with interferon (IFN) signaling, JAK/STAT, and antigen processing/presentation pathways. Aberrant IFN regulation in the setting of AR suggests that tumor cell intrinsic mechanisms have conferred a growth advantage despite the presence of immune-mediated pressure unleashed by PD-1/L1 antibody blockade. Analysis of transcripts progressively upregulated during acquisition of AR in vivo and transcripts upregulated by IFN exposure of isolated AR cell lines in vitro identified the E3 ubiquitin ligase TRIM7 as a candidate mediator of the AR phenotype. A specific pocket on the PRY/SPRY domain of TRIM7 has been shown to bind viral peptides, resulting in ubiquitination and degradation as a mechanism to mitigate viral propagation. Here we describe the evaluation of small molecule inhibitors (SMI) designed to the TRIM7 PRY/SPRY viral-pocket domain, where binding specificity was confirmed using mass spec analysis. These SMI competitively inhibited TRIM7 mediated stabilization of RACO-1, resulting in decreased cancer cell proliferation which was associated with altered c-Jun/AP-1 transcription. TRIM7 SMI also disrupted STING and MAVS ubiquitination and restored IFN responsiveness and sensitivity to PD-1 inhibitor treatment. Finally, given the position of TRIM7 downstream of growth factor receptor and kras signaling, combination of TRIM7 inhibitors with kras, braf, and/or MEK inhibitors was shown to additively suppress proliferation of tumor cells with kras mutations. These results provide evidence that upregulation of TRIM7 mediates both a pro-proliferative and IFN resistant phenotype in the setting of CPI-AR, and that inhibition of TRIM7 with small molecule inhibitors may prevent or reverse AR to PD-1/L1 blockade. Citation Format: Thuy-Ai Tran Nguyen, Marc Morra, Kevin Li, Ulhas Bhatt, Joseph Mwangi, Yisel Rivera-Molina, Nathan Oien, Julio Medina, Taylor H. Schreiber, George Fromm. Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6584.

Abstract 1927: Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors

Abstract More than 90% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS that drive it into an active, GTP-bound state (KRAS(ON)), producing excessive signaling via MAPK and other effector pathways. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). Exposure to RMC-7977 suppressed the MAPK biomarker p-ERK, reduced cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines. Assessment of RMC-7977 in a series of PDAC preclinical model systems revealed broad anti-tumor activity and treatment was well tolerated at translatable doses. The K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) autochthonous mouse model was utilized to study the effects of Ras-GTP inhibition in both tumor and normal tissues. At doses that drove anti-tumor activity, RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissue, with full pathway inhibition at 4 h post treatment that was restored by 24 h. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. RMC-7977 treatment drove regressions and an unprecedented ~3-fold extension of overall survival in KPC mice. After an initial response, all tumors eventually developed resistance on treatment, affording the opportunity to study acquired resistance mechanisms to RAS-GTP inhibition. Strikingly, in all resistant tumors, acute inhibition of MAPK and PI3K signaling was still apparent following treatment. These results indicate that RAS signaling re-activation mechanisms that have been reported as clinical mechanisms of resistance to mutant-selective KRASG12C inhibitors (e.g. second-site mutations or upregulation of upstream RTK) may be insufficient to confer resistance to a compound that inhibits all RAS isoforms. Rather, in this model we observed activation of alternative oncogenic mechanisms downstream of RAS effector signaling, including frequent focal amplification of Myc, focal amplification of Jun, and/or activation of Yap/Taz/Tead signaling in most resistant KPC tumors. Together these results demonstrate preclinical activity of RMC-7977 at tolerable doses, producing unprecedented responses even in highly chemoresistant PDAC models such as the KPC mouse. The observed range of potential resistance mechanisms suggests that RAS-GTP inhibition imposes a narrow evolutionary path to the development of acquired resistance in this context. These preclinical findings may help inform the ongoing development of investigational RAS inhibitors and the design of potential combinatorial treatment strategies to forestall resistance. Citation Format: Urszula N. Wasko, Jingjing Jiang, Yingyun Wang, Lorenzo Tomassoni, Lingyan Jiang, Marie Menard, Alvaro Curiel-Garcia, Tanner C. Dalton, Margo Orlen, Cole Edwards, Clint Stalnecker, Julien Dilly, Stephanie Chang, Stephen A. Sastra, Carmine F. Palermo, Timour Baslan, Sha Tian, Matthew Holderfield, Elsa Quintana, Zhengping Wang, Jacqueline A. Smith, Andrea Califano, David Wildes, Andrew J. Aguirre, Robert H. Vonderheide, Ben Z. Stanger, Scott W. Lowe, Channing J. Der, Mallika Singh, Kenneth P. Olive. Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors [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 1927.

Circulating Osteopontin Predicts Clinical and Radiological Response in First-Line Treatment of Advanced Non-Small Cell Lung Cancer

PurposePembrolizumab-based regimens are conditioned by the expression of PD-L1, but durable response rate is limited by innate and acquired resistance mechanisms. Here, we focus on osteopontin (OPN), an upfront biomarker of senescence, which closely associated with natural history of non-small cell lung cancer (NSCLC).MethodsSeventy-nine patients eligible to pembrolizumab regimens—alone or in combination with chemotherapy—as first-line treatment of advanced NSCLC were enrolled. Predictive value of OPN toward iRECIST progression disease (PD) was set as first outcome. Secondary ones included performance status (ECOG) at baseline, early (first and best) responses, and overall survival (OS).ResultsHigh Serum OPN characterized patients with worse ECOG-PS (p = 0.015) at baseline and subjects experienced PD/death at first (OR 1.17 [1.02 to 1.35]; p = 0.030) and best responses (0.04 [0.00 to 0.81]; p = 0.035). OPN was associated with time-to-progression (B -2.74 [−4.46 to −1.01]) and time-to death (−0.13 [−0.20 to −0.05]). Cox regression models unveil a predictive value for iRECIST-PD (HR 1.01 [1.00 to 1.02]; p = −0.005), RECIST-PD (HR 1.01 [1.00 to 1.02]; p = 0.017), and OS (HR 1.02 [1.01 to 1.03]; p = 0.001). These models were internally validated through bootstrap resampling and characterized by relevant discrimination ability at ROC curve analyses.ConclusionBaseline levels of serum OPN is closely associated with performance status and short/long term outcomes in patients with advanced NSCLC, which are candidate to pembrolizumab-based regimens. As upfront biomarker of senescence, OPN may pave the way for future studies focusing on senescence patterns in NSCLC.

Exploration of novel 6,8,9-trisubstituted purine analogues: synthesis, in vitro biological evaluation, and their effect on human cancer cells.

Cancer, a leading global cause of mortality, demands continuous advancements in therapeutic strategies. This study focuses on the design and synthesis of a novel series of purine derivatives, specifically 6-(substituted phenyl piperazine)-8-(4-phenoxyphenyl)-9-cyclopentyl purine derivatives (5-11). The motivation behind this endeavor lies in addressing acquired resistance mechanisms in cancer cells, a significant hurdle in current treatment modalities. The synthesis, starting from 4,6-dichloro-5-nitropyrimidine, involves a multi-step process, resulting in seven new purine derivatives. Biological evaluation against human liver, colon, and breast cancer cells (Huh7, HCT116, and MCF7, respectively) was performed using the SRB assay. Among the synthesized analogs, compounds 5 and 6, exhibited notable cytotoxic activity, surpassing clinically used positive controls 5-Fluorouracil and Fludarabine in terms of efficacy. This research underscores the potential of purine derivatives with a phenyl group at the C-8 position as a scaffold for developing compounds with improved anticancer properties. The findings offer insights for future exploration and development of novel agents in cancer pharmaceutical research.

Abstract B066: PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer

Abstract Breast cancer (BC) is the most diagnosed cancer in females in the United States and the second leading cause of cancer related deaths among women. The fibroblast growth factor receptor (FGFR) signaling pathway is frequently activated in BC, making it an attractive therapeutic target, especially in metastatic disease. However, clinical trials using FGFR inhibitors in BC have been disappointing in comparison to other cancer types treated with these therapies. As a result, BC patients with activated FGFR do not have a targeted therapeutic option. Failure of FGFR inhibitors is indicative of intrinsic or acquired resistance mechanisms in metastatic BC. Herein we demonstrate significant efficacy of the FGFR inhibitor, pemigatinib, in the 4T07 murine model of BC utilizing a tail vein model to induce pulmonary tumors. Despite this initial response, pemigatinib fails to eliminate minimal residual disease (MRD) and allows for accelerated tumor return after pemigatinib cessation. Characterizing the MRD by IHC following pemigatinib treatment shows that expression of platelet-derived growth factor receptor (PDGFR) is significantly increased on tumor cells. This rise in PDGFR expression following pemigatinib treatment increases the responsiveness of 4T07 cells to platelet-derived growth factor ligand indicating PDGFR may act as an alternate signaling pathway bypassing FGFR signaling. Mechanistically, previous studies indicate that PDGFR protein expression is transcriptionally repressed by restriction of enhancer positioning by the CTCF insulator. DNA methylation surrounding CTCF causes its dissociation enhancing protein expression of PDGFR. Along these lines cotreatment with a DNA methyl transferase inhibitor prevents the increase of PDGFR protein expression induced by pemigatinib. Overall, our study determined that a highly potent FGFR inhibitor can inhibit tumor growth in a metastatic site, but it fails to eliminate MRD giving rise to tumor recurrence. Upregulation of PDGFR may act as a bypass mechanism of resistance allowing for tumor cell survival during pemigatinib treatment. Finally, our findings suggest that dual inhibition of DNA methylation and FGFR could improve patient response in metastatic BC. Citation Format: Mitchell Ayers, Michael Wendt. PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B066.

Abstract A022: Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA

Abstract Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer. A key driver of this near universal lethality is SCLC’s recalcitrance to therapy. The standard of care for SCLC is a chemotherapy doublet of etoposide and cis/carboplatin in combination with immunotherapy (EP+IO). While most tumors respond to these therapies, >80% of SCLCs progress within one year of treatment. Additionally, the aggressive clinical course of SCLC has historically precluded collection of patient-matched treatment naïve and recurrent tumor tissue traditionally required to study evolution. In lieu of tissue, several groups have used circulating tumor DNA (ctDNA) to study SCLC evolution. However, these studies used targeted sequencing panels that analyze <0.04% of the SCLC genome. Using new methods to comprehensively trace how SCLCs rapidly evolve to therapy resistance is a significant unmet need. Methods: Medical records were reviewed to identify treatment naïve SCLC patients treated at MD Anderson that had both pre- and post-EP+IO liquid biopsies collected under IRB approved protocols. ctDNA was isolated from plasma using a MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Bio). Germline DNA was extracted from buffy coat samples using a QIAamp DNA Blood Midi Kit (Qiagen). Deep whole genome sequencing (WGS) (>100X) of ctDNA and WGS of germline DNA (>60X) was performed using an Illumina NovaSeq X sequencer. Reads were aligned to the hg38 reference genome using BWA-mem. Somatic mutations were called using MuTect2, and clonal and subclonal copy number alterations were identified using Battenberg. DPClust was used to reconstruct cancer cell populations, leveraging mutation and copy number calls. Results: We identified several deleterious genomic alterations impacting TP53 and RB1 tumor suppressor genes, known genetic drivers of SCLC development. We also detected recurrent, whole chromosome arm loss of heterozygosity events impacting many chromosomes in treatment naïve samples, some of which are known to be present in >80% of SCLCs. Additionally, we found that both treatment naïve and recurrent SCLCs were composed of multiple subclones, each defined by dozens to thousands of unique somatic mutations. COSMIC signature analysis identified high activity of tobacco carcinogens, APOBEC, and replicative-based processes across ctDNA samples. We also identified emergence of novel subclones carrying unique copy number alterations following EP+IO recurrence. Importantly, the affected regions encode key effectors that regulate anti-tumor immune responses and cell survival following DNA damage, both of which are intimately related to the mechanism of action of EP+IO. Conclusions: Our data shows that deep WGS of ctDNA recapitulates known genetics of SCLC and allows for sensitive characterization of therapy resistant clones. Using this method, we have captured snapshots of both intrinsic and acquired resistance mechanisms to frontline EP+IO. Deep WGS of ctDNA is a promising approach to comprehensively dissect routes of SCLC evolution and therapy resistance. Citation Format: Benjamin B. Morris, Zhihui Zhang, Simon Heeke, Kyle Concannon, Bingan Zhang, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, J. Jack Lee, Jianjun Zhang, Don L. Gibbons, Ara A. Vaporciyan, Carl M. Gay, Hai T. Tran, John V. Heymach, Lauren A. Byers, Peter Van Loo. Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A022.

PREVALENCE OF ANTIBIOTIC RESISTANCE IN PSEUDOMONAS AERUGINOSA

One of the main bacteria responsible for hospital-acquired illnesses is Pseudomonas aeruginosa. Through chromosomal changes or the horizontal acquisition of resistant determinants, antibiotic resistance can be easily developed it. High-risk clones, like ST175, are spreading together with the rising frequency of extensively-drug-resistant (XDR) or multi-drug-resistant (MDR) P. aeruginosa isolates. MDR/XDR infections should be taken seriously since they can make it difficult to choose the best empirical and conclusive antimicrobial therapies. New avenues for the treatment of MDR/XDR P. aeruginosa infections have been opened by the introduction of powerful new antibiotics. Pseudomonas aeruginosa strains are known to withstand the majority of antibiotics by utilizing their high levels of intrinsic and acquired resistance mechanisms. Furthermore, recalcitrance and infection recurrence are caused by P. aeruginosas adaptive antibiotic resistance, a recently identified process that combines biofilm-mediated resistance and the development of multidrug-tolerant persister cells. There is a growing need for and interest in the research and development of alternative therapeutic approaches that offer fresh approaches to combat P. aeruginosa infections. Much recent research has documented various novel therapeutic methods that have proven pronouncedly effective in treating drug-resistant P. aeruginosa strains, but largely at the preclinical stages. This review focuses on the Prevalence of antibiotic resistance in Pseudomonas aeruginosaand also provides an overview of the characteristics of pseudomonas bacteria, various infections caused by them, the mechanism of antibiotic resistance, their clinical implications, Challenges in treatment, strategies for management and control, and future perspectives and research directions.

Potentiation of Antibiotic Activity of Aztreonam against Metallo-β-Lactamase-Producing Multidrug-Resistant Pseudomonas aeruginosa by 3-O-Substituted Difluoroquercetin Derivatives.

The combination of aztreonam (ATM) and ceftazidime-avibactam (CAZ-AVI; CZA) has shown therapeutic potential against serine-β-lactamase (SBL)- and metallo-β-lactamase (MBL)-producing Enterobacterales. However, the ability of CZA to restore the antibiotic activity of ATM is severely limited in MBL-producing multidrug-resistant (MDR) Pseudomonas aeruginosa strains because of the myriad of intrinsic and acquired resistance mechanisms associated with this pathogen. We reasoned that the simultaneous inhibition of multiple targets associated with multidrug resistance mechanisms may potentiate the antibiotic activity of ATM against MBL-producing P. aeruginosa. During a search for the multitarget inhibitors through a molecular docking study, we discovered that di-F-Q, the previously reported efflux pump inhibitor of MDR P. aeruginosa, binds to the active sites of the efflux pump (MexB), as well as various β-lactamases, and these sites are open to the 3-O-position of di-F-Q. The 3-O-substituted di-F-Q derivatives were thus synthesized and showed hereto unknown multitarget MDR inhibitory activity against various ATM-hydrolyzing β-lactamases (AmpC, KPC, and New Delhi metallo-β-lactamase (NDM)) and the efflux pump of P. aeruginosa, presumably by forming additional hydrophobic contacts with the targets. The multitarget MDR inhibitor 27 effectively potentiated the antimicrobial activity of ATM and reduced the MIC of ATM more than four-fold in 19 out of 21 MBL-producing P. aeruginosa clinical strains, including the NDM-producing strains which were highly resistant to various combinations of ATM with β-lactamase inhibitors and/or efflux pump inhibitors. Our findings suggest that the simultaneous inhibition of multiple MDR targets might provide new avenues for the discovery of safe and efficient MDR reversal agents which can be used in combination with ATM against MBL-producing MDR P. aeruginosa.

First-308: Phase III study of tinengotinib versus physician's choice in patients with FGFR-altered, chemotherapy- and FGFR inhibitor–refractory/relapsed cholangiocarcinoma.

TPS575 Background: Fibroblast growth factor (FGFR) alterations occur in 10-15% of adult patients with advanced intrahepatic cholangiocarcinoma (CCA) and 1-2% of adult patients with advanced extrahepatic cholangiocarcinoma. The first generation FGFR inhibitors (FGFRi) pemigatinib and futibatinib, have been approved for the treatment of advanced CCA with FGFR2 fusions or rearrangements after systemic chemotherapy. However, disease progression occurs within 6-9 months. Secondary polyclonal mutations in the FGFR kinase domain represent a prominent acquired resistance mechanism. Tinengotinib, a novel multi-kinase inhibitor with high potency against a variety of FGFR2 kinase domain mutations, has shown promising clinical benefit in subjects with FGFR-altered metastatic CCA who were heavily pretreated with chemotherapy and FGFRi(s) in phase I/II clinical trials. (NCT03654547, NCT04742959, NCT04919642). Methods: FIRST-308 is a phase III, randomized, global multicenter study to evaluate the efficacy and safety of oral tinengotinib versus Physician’s Choice in subjects with FGFR-altered, chemotherapy- and FGFRi-refractory/relapsed CCA. Approximately 200 subjects will be enrolled in US, Europe, and Asia. Key eligibility criteria include age ≥ 18 years, ECOG performance status 0 or 1, documentation of FGFR2 fusion/rearrangement gene status, prior treatment with at least one line of chemotherapy and exactly one prior FDA-approved FGFRi for unresectable or metastatic disease. The study includes Part A and Part B. The Part A is to select a dose for Part B. Eligible subjects will be randomized in a 2:2:1 ratio to receive tinengotinib 8 mg QD, tinengotinib 10 mg QD or Physician's Choice (FOLFOX or FOLFIRI) in Part A or 2:1 in Part B to receive the recommended Part B dose or Physician's Choice. The stratification factors at randomization include geographic region, prior lines of chemotherapy (1 or ≥ 2) and Physician’s choice. Tinengotinib will be administered orally QD in 28-day cycles. Subjects will continue to receive study treatment until confirmed disease progression, unacceptable toxicity, withdrawal of consent, or termination of study by Sponsor, whichever occurs first. For Part A, the primary endpoint is safety/tolerability; secondary endpoints include objective response rate (ORR), duration of response (DOR) and PK analysis. For Part B, the primary endpoint is progression-free survival (PFS); secondary endpoints include overall survival (OS), ORR, DOR, safety, quality of life and population PK. Exploratory endpoints will assess the correlations between baseline FGFR2 alterations by cfDNA and efficacy, and exposure-response in terms of efficacy and safety. Study is open for enrollment. Clinical trial information: NCT05948475 .

Next-generation sequencing reveals genetic heterogeneity and resistance mechanisms in patients with EGFR-mutated non-small cell lung cancer treated with afatinib.

Afatinib, an irreversible ErbB family inhibitor, is widely used as first-line treatment in advanced lung adenocarcinoma patients harbouring mutant epidermal growth factor receptor (EGFR). With the advancements in next-generation sequencing (NGS), comprehensive research into the clinical impact of co-occurring genetic mutations and the molecular mechanisms of acquired resistance is required for afatinib users. From January 2010 to December 2019, we enrolled patients with advanced lung adenocarcinoma with EGFR mutations using afatinib as first-line treatment, and we retrospectively collected pre- and post-afatinib treatment specimens from these patients for NGS testing. Of the 362 enrolled patients, 73 samples (68.9%) from 56 patients successfully returned complete NGS reports. In pre-afatinib treatment specimens, the most frequent co-occurring alterations were TP53, MUC16, USH2A, SNYE1, RECQL4 and FAT1; however, they were not related to progression-free survival. Small cell lung cancer transformation, EGFR p.T790M, amplification of MET, ERBB2, KRAS, EGFR, cell cycle-regulated genes and MDM2, and PTEN alterations were identified as acquired resistance mechanisms. EGFR p.T790M (p=0.0304) and APC alterations (p=0.0311) in post-afatinib specimens were significantly associated with longer overall survival, while MET amplification was significantly associated with poor overall survival (p=0.0324). The co-occurrence of TP53 alterations was significantly associated with shorter overall survival (p=0.0298). Our results show that the frequent co-occurring alterations in advanced EGFR-mutated lung adenocarcinoma did not influence the effectiveness of afatinib. EGFR p.T790M is not only the major resistance mechanism to afatinib but also related to favourable survival outcomes. MET amplification and TP53 mutations were associated with poorer overall survival.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data.ResultsWe found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells.ConclusionOverall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Cereblon-based Bifunctional Degrader of SOS1, BTX-6654, Targets Multiple KRAS Mutations and Inhibits Tumor Growth.

Mutations within the oncogene KRAS drive an estimated 25% of all cancers. Only allele-specific KRAS G12C inhibitors are currently available and are associated with the emergence of acquired resistance, partly due to upstream pathway reactivation. Given its upstream role in the activation of KRAS, son of sevenless homolog 1 (SOS1), has emerged as an attractive therapeutic target. Agents that target SOS1 for degradation could represent a potential pan-KRAS modality that may be capable of circumventing certain acquired resistance mechanisms. Here, we report the development of two SOS1 cereblon-based bifunctional degraders, BTX-6654 and BTX-7312, cereblon-based bifunctional SOS1 degraders. Both compounds exhibited potent target-dependent and -specific SOS1 degradation. BTX-6654 and BTX-7312 reduced downstream signaling markers, pERK and pS6, and displayed antiproliferative activity in cells harboring various KRAS mutations. In two KRAS G12C xenograft models, BTX-6654 degraded SOS1 in a dose-dependent manner correlating with tumor growth inhibition, additionally exhibiting synergy with KRAS and MEK inhibitors. Altogether, BTX-6654 provided preclinical proof of concept for single-agent and combination use of bifunctional SOS1 degraders in KRAS-driven cancers.