Adaptive Resistance Mechanisms Research Articles

Modulation of the Vascular-Immune Environment in Metastatic Cancer

Simple SummaryAbnormal blood vessels restrict immune cell access into primary cancers and thus contribute to resistance to immunotherapy. In contrast, vessel remodeling or normalization strategies increase anti-cancer immune responses. Much less is known about the inter-relationship of blood vessels and immune cells at secondary metastatic sites. This review specifically explores how abnormal vascular features in the pre-metastatic niche can be targeted and potentially repaired. Moreover, the growth of established metastases relies on blood supply from both pre-existing, co-opted and newly formed angiogenic vessels. The co-existence of blood vessels with different maturation status may impact on vascular targeting strategies and responsiveness to immunotherapy, thus requiring innovative approaches to treat advanced cancer.Advanced metastatic cancer is rarely curable. While immunotherapy has changed the oncological landscape profoundly, cure in metastatic disease remains the exception. Tumor blood vessels are crucial regulators of tumor perfusion, immune cell influx and metastatic dissemination. Indeed, vascular hyperpermeability is a key feature of primary tumors, the pre-metastatic niche in host tissue and overt metastases at secondary sites. Combining anti-angiogenesis and immune therapies may therefore unlock synergistic effects by inducing a stabilized vascular network permissive for effector T cell trafficking and function. However, anti-angiogenesis therapies, as currently applied, are hampered by intrinsic or adaptive resistance mechanisms at primary and distant tumor sites. In particular, heterogeneous vascular and immune environments which can arise in metastatic lesions of the same individual pose significant challenges for currently approved drugs. Thus, more consideration needs to be given to tailoring new combinations of vascular and immunotherapies, including dosage and timing regimens to specific disease microenvironments.

Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma.

Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCβ and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

Treatment strategies for cryptococcal infection: challenges, advances and future outlook.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.

Downregulation of lncRNA TSLNC8 promotes melanoma resistance to BRAF inhibitor PLX4720 through binding with PP1α to re-activate MAPK signaling.

Approximately 60% of patients with melanoma harbor BRAF mutation and targeting BRAF offers enormous advance in the treatment of those patients. Unfortunately, the efficacy of the BRAF inhibitors is usually restricted by the onset of drug resistance. Therefore, better understanding of the adaptive drug resistance mechanisms is essential for the development of alternative therapeutic strategies, and offers more promising measures to promote the short duration of response to BRAF inhibitors. The levels of tumor suppressive long noncoding RNA on chromosome 8p12 (TSLNC8) were evaluated by qPCR. The MTT assay, colony formation assay, apoptosis assay, and in vivo xenograft tumor model were performed to assess the functions of TSLNC8 on drug resistance. Western blotting, RNA pull-down, and RNA immunoprecipitation (RIP) assays were applied to investigate the mechanisms of TSLNC8 in melanoma. Herein, our findings demonstrate that TSLNC8 is significantly downregulated in BRAF inhibitor-resistant melanoma tissues and cells. Moreover, downregulation of TSLNC8 in BRAF inhibitor sensitive cells reduces the toxicity response to BRAF inhibitor PLX4720, and inhibits apoptosis of melanoma cells-treated with PLX4720. Further assay elucidates that TSLNC8 can bind with the catalytic subunit of protein phosphatase 1α (PP1α) to regulate its distribution, and Downregulation of TSLNC8 results in PP1α cytoplasmic accumulation, thus re-activating the MAPK signaling. Eventually, the overexpression of TSLNC8 in BRAF inhibitor PLX4720-resistant melanoma cells restores the sensitive to BRAF inhibitor. Collectively, our research provides a compelling rationale for resistance to BRAF inhibitor in melanoma, and the patient might benefit from the combinatorial therapy of BRAF inhibitors and lncRNA TSLNC8.

Changes in the Tumor Immune Microenvironment during Disease Progression in Patients with Ovarian Cancer.

Simple SummaryImmunotherapy has been a successful treatment for many cancers. However, no immunotherapy treatment has been approved for ovarian cancer due to low efficacy in this patient group. This study investigated the cellular and molecular changes from primary ovarian tumors, at the time of diagnosis, to recurrence, where the disease returns after surgery and chemotherapies. Here we examined the immune contexture to better understand subdued responses to immunotherapy and identify additional, potentially complimentary, therapeutic targets. Indications of the development of adaptive immune resistance during disease progression were observed, with increases in immune and stromal cell infiltration accompanied by the expression of immune suppressive markers. We observed high gene expression of the immune checkpoint genes LAG3 and HAVCR2 (TIM3) in most tumors and increased expression of the immune checkpoint genes TIGIT and CTLA4 in recurrent tumors, compared to the primaries. These markers may be potential candidates for immunotherapy targeting in ovarian cancer.Anti-PD1/PDL1 therapy has proven efficacious against many cancers but only reached modest objective response rates against recurrent ovarian cancer. A deeper understanding of the tumor microenvironment (TME) may reveal other immunosuppressive mechanisms that warrant investigation as immunotherapeutic targets for this challenging disease. Matched primary and recurrent tumors from patients with high-grade serous ovarian carcinoma (HGSC) were analyzed by multicolor immunohistochemistry/immunofluorescence for the presence of T cells, B cells, macrophages, and for the expression of immunosuppressive and HLA molecules. Cancer- and immune-related gene expression was assessed by NanoString analysis. Recurrent tumors showed increased infiltration by immune cells, displayed higher expression of PDL1, IDO, and HLA molecules, and contained more stromal tissue. NanoString analysis demonstrated increased expression of gene signatures related to chemokines and T cell functions in recurrent tumors. The ovarian tumors showed high gene expression of LAG3 and HAVCR2 (TIM3) and enhanced levels of TIGIT and CTLA4 in recurrent tumors compared to primary tumors. The majority of HGSC developed into a more inflamed phenotype during progression from primary to recurrent disease, including indications of adaptive immune resistance. This suggests that recurrent tumors may be particularly sensitive to inhibition of adaptive immune resistance mechanisms.

Tackling Pseudomonas aeruginosa Virulence by Mulinane-Like Diterpenoids from Azorella atacamensis.

Pseudomonas aeruginosa is an important multidrug-resistant human pathogen by dint of its high intrinsic, acquired, and adaptive resistance mechanisms, causing great concern for immune-compromised individuals and public health. Additionally, P. aeruginosa resilience lies in the production of a myriad of virulence factors, which are known to be tightly regulated by the quorum sensing (QS) system. Anti-virulence therapy has been adopted as an innovative alternative approach to circumvent bacterial antibiotic resistance. Since plants are known repositories of natural phytochemicals, herein, we explored the anti-virulence potential of Azorella atacamensis, a medicinal plant from the Taira Atacama community (Calama, Chile), against P. aeruginosa. Interestingly, A. atacamensis extract (AaE) conferred a significant protection for human lung cells and Caenorhabditis elegans nematodes towards P. aeruginosa pathogenicity. The production of key virulence factors was decreased upon AaE exposure without affecting P. aeruginosa growth. In addition, AaE was able to decrease QS-molecules production. Furthermore, metabolite profiling of AaE and its derived fractions achieved by combination of a molecular network and in silico annotation allowed the putative identification of fourteen diterpenoids bearing a mulinane-like skeleton. Remarkably, this unique interesting group of diterpenoids seems to be responsible for the interference with virulence factors as well as on the perturbation of membrane homeostasis of P. aeruginosa. Hence, there was a significant increase in membrane stiffness, which appears to be modulated by the cell wall stress response ECFσ SigX, an extracytoplasmic function sigma factor involved in membrane homeostasis as well as P. aeruginosa virulence.

Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2–10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

Structural and Functional Diversity of Resistance–Nodulation–Cell Division Transporters

Multidrug resistant (MDR) bacteria are a global threat with many common infections becoming increasingly difficult to eliminate. While significant effort has gone into the development of potent biocides, the effectiveness of many first-line antibiotics has been diminished due to adaptive resistance mechanisms. Bacterial membrane proteins belonging to the resistance-nodulation-cell division (RND) superfamily play significant roles in mediating bacterial resistance to antimicrobials. They participate in multidrug efflux and cell wall biogenesis to transform bacterial pathogens into "superbugs" that are resistant even to last resort antibiotics. In this review, we summarize the RND superfamily of efflux transporters with a primary focus on the assembly and function of the inner membrane pumps. These pumps are critical for extrusion of antibiotics from the cell as well as the transport of lipid moieties to the outer membrane to establish membrane rigidity and stability. We analyze recently solved structures of bacterial inner membrane efflux pumps as to how they bind and transport their substrates. Our cumulative data indicate that these RND membrane proteins are able to utilize different oligomerization states to achieve particular activities, including forming MDR pumps and cell wall remodeling machineries, to ensure bacterial survival. This mechanistic insight, combined with simulated docking techniques, allows for the design and optimization of new efflux pump inhibitors to more effectively treat infections that today are difficult or impossible to cure.

A Multi-Center, Dose-Finding Study to Assess Safety, Tolerability, Pharmacokinetics and Preliminary Efficacy of a Novel IRAK4 Inhibitor CA-4948 in Combination with Ibrutinib, in Patients with Relapsed or Refractory Hematologic Malignancies

A Multi-Center, Dose-Finding Study to Assess Safety, Tolerability, Pharmacokinetics and Preliminary Efficacy of a Novel IRAK4 Inhibitor CA-4948 in Combination with Ibrutinib, in Patients with Relapsed or Refractory Hematologic Malignancies

Safety, Pharmacokinetics and Activity of CA-4948, an IRAK4 Inhibitor, for Treatment of Patients with Relapsed or Refractory Hematologic Malignancies: Results from the Phase 1 Study

Introduction CA-4948 is a novel oral small molecule inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4). IRAK4 is part of the Myddosome signaling pathway, and is essential for signaling downstream of toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family in immune cells including B lymphocytes. . Dysregulated signaling in these pathways is frequently observed in Non-Hodgkin Lymphomas (NHL), particularly in ABC-subtype of Diffuse Large B-Cell Lymphoma (DLBCL) and Waldenström macroglobulinemia (WM)[1]. Furthermore, IRAK4 activation has recently been identified as a driver of an adaptive resistance mechanism of malignancies [2]. CA-4948 is the first clinical candidate targeting IRAK4 to be evaluated in cancer patients. Here we present Phase I results of CA-4948 in patients with relapsed/refractory hematologic malignancies. Method CA-4948-101 is a phase I trial dose escalation trial with a 3 + 3 design. Seven dosing cohorts were assessed including 50 and 100 mg daily (QD), and 50, 100, 200, 300, or 400 mg twice daily (BID) continuous oral monotherapy in 21-day cycles. Objectives included safety and tolerance (primary), pharmacokinetic (PK), pharmacodynamics (PD) and early efficacy (secondary), and biomarker correlations (exploratory). As of July 2020, 30 patients with relapsed or refractory, NHL have been enrolled, including DLBCL (n=14), FL (n=6), transformed HGBL (n=1), WM (n=3), LPL (n=2), MZL (n=2) and MCL (n=2). Median age is 68.5 (range 50 - 87; 5 female / 25 male). Median number of prior treatments is 4 (range 1 - 8, incl. 5 CAR-T-cell or ASCT treatments). Results CA-4948 has been generally well tolerated. Eight patients were exposed at the highest dose level of 400 mg BID: 2 of 5 DLT-evaluable patients had Grade 3 rhabdomyolysis (DLTs), without complications and reversible after treatment interruption and hydration / analgesic treatment - both subsequently continued treatment at lower doses of 200 or 300 mg BID, respectively. Six patients have tolerated 300 mg BID well without DLT. Most non-hematologic TEAEs were Grade 1 or 2 and manageable, including diarrhea, vomiting, fatigue, dyspnea, and myalgia. Hematologic TEAEs including neutropenia, anemia, thrombocytopenia was mild/moderate and without complications. There were only 4 Grade 3 combined episodes in 18 patients at dose levels ranging between 200 and 400 mg BID without complications (Table 1). Drug discontinuation occurred in only 3 cases for rash, fatigue, and asymptomatic amylase/lipase elevation at lower dose levels. No toxic deaths occurred. Favorable and predictable PK characteristics included rapid absorption with Tmax at 2-3 hours, T1/2 of 6 hours (supporting bid dosing), moderate accumulation, and dose-proportional increases in exposure. Dose proportional PD changes were shown for inflammatory cytokine reductions. Treatment duration ranged between 15 days and 19+ months with sustained disease control. Eight of 28 evaluable patients experienced overall tumor burden decreases of ≥20% from baseline - more at higher doses (Table 2). A WM patient with 6 prior lines of treatment, sustained PR underwent intra-patient dose escalation and had a dose/response relationship and very good treatment tolerance (Figure 1). The median duration at doses of 200 or 300 mg bid was >6 months (range 1-19+; including 3 cases of 11, 14, and 19+ mo.). Downstream pharmacodynamic markers of IRAK4 and molecular characteristics including cell-of-origin will be presented. Conclusion CA-4948 demonstrates good safety profile, desirable pharmacokinetic properties, and preliminary clinical activity. Enrollment is ongoing to support the anticipated RP2D of 300 mg bid. These encouraging results provide clinical validation of IRAK4 as a viable, novel therapeutic target in B-cell malignancies will lead to clinical combinations with synergistic drugs including BTK or BCL2 inhibitors [3] in patients with advanced hematological malignancies. Clinical trial: NCT03328078.

Abstract B01: Genome-scale shRNA screen provides insight into the role of CUL3 in adaptive mechanisms of resistance to mutant BRAF inhibition

Abstract MAPK inhibition with targeted BRAF inhibitors has revolutionized the treatment of BRAF-mutant melanoma. However, in most cases, the effects of mutant BRAF inhibition are transient, owing to the acquisition of drug resistance. To resolve mechanisms that drive resistance to mutant BRAF inhibition, we performed a genome-scale shRNA screen capable of revealing genes whose loss of function confers resistance to vemurafenib (VEM). We validated ten of our top candidates with independent lentiviral shRNA constructs. We found that knockdown of NF1 and CUL3 confers resistance to VEM. This result is consistent with previous genome-scale loss-of-function screens that have also identified NF1 and CUL3 as drivers of resistance to mutant BRAF inhibition. NF1 encodes a protein that hydrolyzes Ras-GTP. Not surprisingly, it has been shown that loss of NF1 results in increased Ras-GTP levels that lead to reactivation of MAPK in the presence of VEM. However, little is known about the mechanistic changes that underlie CUL3-mediated resistance. Here, we show that unlike the classical reactivation of MAPK that is seen with the loss of NF1, loss of CUL3 results in increased signaling through a Rac1-dependent focal-adhesion-like process that is accompanied by sustained MAPK activity in the presence of VEM. Furthermore, we show that this mechanism of resistance can be abrogated by inhibiting the Src kinase family, a nine-member protein family that critically mediates integrin signaling. In relation to other known drivers of resistance to mutant BRAF inhibition, loss of NF1 reactivates MAPK in a manner much like RAS mutations or BRAF alterations/amplification. By contrast, loss of CUL3 leads to resistance reminiscent of a reversible therapy-resistant state that lacks the key genomic alterations in MAPK signaling proteins. Citation Format: Marion Vanneste, Eliot Zhu, Charlotte Feddersen, Afshin Varzavand, Tyler Foley, Lei Zhao, Rob Piper, Christopher Stipp, Adam Dupuy, Michael Henry. Genome-scale shRNA screen provides insight into the role of CUL3 in adaptive mechanisms of resistance to mutant BRAF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B01.

CD44/HA signaling mediates acquired resistance to a PI3K\u03b1 inhibitor

Most luminal breast carcinomas (BrCas) bearing PIK3CA mutations initially respond to phosphoinositide-3-kinase (PI3K)-α inhibitors, but many eventually become resistant. The underlying mechanisms of this resistance remain obscure. In this work, we showed that a CD44high state due to aberrant isoform splicing was acquired from adaptive resistance to a PI3Kα inhibitor (BLY719) in luminal BrCas. Notably, the expression of CD44 was positively correlated with estrogen receptor (ER) activity in PIK3CA-mutant breast cancers, and ER-dependent transcription upon PI3Kα pathway inhibition was in turn mediated by CD44. Furthermore, the interaction of CD44 with the ligand hyaluronan (HA) initiated the Src-ERK signaling cascade, which subsequently maintained AKT and mTOR activity in the presence of a PI3Kα inhibitor. Activation of this pathway was prevented by disruption of the CD44/HA interaction, which in turn restored sensitivity to BLY719. Our results revealed that an ER-CD44-HA signaling circuit that mediates robust compensatory activation of the Src-ERK signaling cascade may contribute to the development of acquired resistance to PI3Kα inhibitors. This study provides new insight into the mechanism of adaptive resistance to PI3Kα inhibition therapy.

Endothelin-1 axis fosters YAP-induced chemotherapy escape in ovarian cancer.

The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, β-arrestin1 (β-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ETAR/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients.

Abstract LB-021: Focal adhesive kinase inhibitor IN10018 sensitizes KRAS mutant cancer and overcomes drug resistance of KRAS G12C inhibition

Abstract KRAS mutation is found in around 20-30% of human cancer. Previously, lack of targeting therapy limited the treatment outcomes. Recently, KRAS G12C inhibitors like AMG510, MRTX849, and MRTX1257 were developed and exhibited promising therapeutic outcomes targeting the mutation. However, a considerable amount of cases showed less sensitivity, indicating either intrinsic or adaptive resistance mechanisms. Other reports and our own data exhibited that Focal Adhesive Kinase (FAK) inhibition showed good treatment effects to KRAS mutant cancer. Interestingly, TCGA data analysis reveal that FAK mRNA expression level is significantly higher in KRAS mutant tumors. Considering that FAK is one predominant target for cancer treatment resistance, we hypothesize that FAK is one of mediator leading to resistance mechanism of KRAS G12C inhibition. To explore our hypothesis, we employed high potent FAK inhibitor IN10018, which is being tested in phase I clinical trial for different cancer types. First, we found that IN10018 is active in a series of KRAS mutated cancer xenograft models. The compound sharply decreased the level of phosphoFAK Y397, a direct marker for FAK activity. Then we investigated whether combination of IN10018 with KRAS G12C inhibitors can show synergic effects in vitro and in vivo. IN10018 in combination with either AMG510 or MRTX1257 showed better effects in killing KRAS G12C cancer cells than each single treatment. More importantly a significant synergy between IN10018 with KRAS G12C inhibitors against KRAS mutated cancer cell lines was observed in vivo. Further evidence showed that KRAS G12C inhibition can significantly increase the expression of phosphoFAK Y397, suggesting a possible underlying mechanism of resistance to KRAS G12C inhibitors. IN10018 significantly decreased the upregulated FAK Y397 phosphorylation in the combination treatments, underlying the mechanism of synergy between the two inhibitors. Our results indicate that KRAS G12C cancer patients can be better treated by the combination of IN10018 with KRAS G12C inhibitor. Citation Format: Baoyuan Zhang, Yan Zhang, Jiangwei Zhang, Ping Liu, Bo Jiao, Zaiqi Wang, Ruibao Ren. Focal adhesive kinase inhibitor IN10018 sensitizes KRAS mutant cancer and overcomes drug resistance of KRAS G12C inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-021.

Abstract 62: Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies

Abstract Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to the destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. Durable control of disease in vivo can be achieved with combinations of targeted therapy with an MCL-1 inhibitor. However, consistent with an adaptive resistance mechanism we have identified, the timing of drug treatment strongly influences the efficacy of the combination therapy. Collectively, our results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses. Our data indicate that optimal sequencing of MCL-1 inhibitors with targeted therapies could overcome widespread and clinically important therapeutic resistance, and achieve prolonged tumor responses in solid tumors. Citation Format: Joan Montero, Cecile Gstalder, Daniel J. Kim, Dorota Sadowicz, Wayne Miles, Michael Manos, Justin R. Cidado, J. Paul Secrist, Adriana E. Tron, Keith Flaherty, F. Stephen Hodi, Charles H. Yoon, Anthony Letai, David E. Fisher, Rizwan Haq. Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 62.

Abstract 1690: Blockade of antitumor milieu-induced tumor cell PIM2 expression potently synergizes with anti-PD-L1 therapy in hepatocellular carcinoma

Abstract Background & Aims: Despite being a typical oncogene, PIM kinase has been reported to suggest a favorable prognosis in some solid tumors, but no research has yet explored the underlying mechanisms. Furthermore, an inconsistent prognosis also hinders the clinical application of PIM inhibitors. We aimed to explore the clinical significance and regulatory mechanism of PIM2, as well as the potential clinical application of PIM2 inhibitor in hepatocellular carcinoma (HCC). Methods: PIM2 expression was assessed in 464 HCC patients who underwent curative resection, and its correlations with prognosis, immune status, and tumor infiltrating leukocytes (TILs) were investigated. Moreover, the underlying regulatory mechanisms of PIM2 and the clinical application of PIM2 inhibitor were also studied. Results: We found that PIM2 primarily displayed an oncogenic function in HCC. However, elevated PIM2 expression in tumor cells predicted a favorable prognosis. Mechanistically, high PIM2 expression in tumors was associated with an adaptive immune response elicited by TILs, and IL-1β, together with TNF-α and IFN-γ, derived from TILs induced the expression of PIM2 by activating the p38/Erk/MAPK and NF-κB signaling pathways in tumor cells. Specifically, high PIM2 expression in tumors was significantly correlated with a high density of T cells and monocytes, and activated T cell-secreting IFN-γ-polarized monocytes induced the tumor cell PIM2 expression in the tumor milieu. Moreover, blockade of antitumor milieu-induced tumor cell PIM2 expression potently augmented the therapeutic efficacy of anti-PD-L1 therapy in HCC. Conclusions: Our study provides important insights into a possible adaptive immune resistance mechanism by which the antitumor milieu induces oncogenic PIM2 expression in tumor cells. Moreover, specific inhibition of PIM2 in tumor cells augments the therapeutic efficacy of anti-PD-L1 therapy in HCC. Citation Format: Juncheng Wang, Shi-Xun Lu, Jinbin Chen, Dongping Chen, Minshan Chen, Zhongguo Zhou. Blockade of antitumor milieu-induced tumor cell PIM2 expression potently synergizes with anti-PD-L1 therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1690.

Abstract 2270: RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells

Abstract Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competent antibodies, RG7769 harbors a PG LALA containing heterodimeric KiH IgG1 Fc-region rendering the BsAb refractory to drug shaving by FcgR-expressing macrophages in the TME, while retaining IgG-pharmacokinetics. RG7769 binds to PD-1 with higher affinity than pembrolizumab and nivolumab. X-ray crystallography demonstrated that the humanized PD-1 binding Fab recognizes a unique glycosylated epitope on PD-1, and potently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions in both biochemical and reporter cell line assays. The humanized TIM-3 binding arm was identified for maximal functional activity using mixed lymphocyte reaction (MLR) assays. Compared with bivalent TIM-3 antibodies, RG7769 shows reduced binding to TIM-3+ myeloid and NK cells, but binds preferentially to dysfunctional T cells expressing PD-1 or both PD-1 and TIM-3, like tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. By virtue of its monovalency, RG7769 induced low antibody internalization on activated T cells when compared with bivalent TIM-3 antibodies, overcoming a major cellular sink for TIM-3 antibodies. In functional assays, RG7769 showed increased IFN-γ secretion by in vitro generated tumor-specific T-cells, increased ex vivo tumor-specific effector functions of T cells from PBMCs of melanoma patients, and enhanced the anti-tumor-activity of TILs from melanoma patients when compared to the monospecific parental PD-1 antibody. Finally, RG7769 showed superior efficacy in controlling s.c. MC38 tumor growth in huPD-1/huTIM-3 transgenic C57/BL6 mice compared to the parental PD-1 antibody. In summary, these preclinical data support the use of RG7769 as a monotherapy and as combination partner for the treatment of patients with solid/hematological tumors. A phase I study is currently ongoing in patients with advanced metastatic solid tumors (NCT03708328). Citation Format: Laura Laura Codarri Deak, Stefan Seeber, Mario Perro, Patrick Weber, Laura Lauener, Standford Chen, Sonja Offner, Stefan Dengl, Friederike Hesse, Adrian Zwick, Marco Boettger, Alexander Bujotzek, Jörg Benz, Guy Georges, Georg Fertig, Valeria Lifke, Jens Fischer, Stephane Leclair, Victor Levitsky, Marta Canamero, Juha Lindner, Sara Colombetti, Stefanie Bendels, Christophe Boetsch, Matthias Fueth, Merlind Muecke, Henry Kao, Pablo Umana, Christian Klein. RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2270.

Abstract 3410: Adaptive resistance mechanisms to KRASG12C specific inhibitors

Abstract Covalent inhibitors of KRASG12C mutants allow specific targeting of KRAS driven signaling yet early studies suggest bypass signaling as a mechanism of resistance. Understanding adaptive signaling mechanisms enabling cells to survive KRASG12C inhibition could identify combination therapies and associated predictive biomarkers. We employed KRASG12C mutant lung cancer cell lines and evaluated their sensitivity in 2D cell culture to mimic the resistant state. We combined small molecule targeted agents (N=292 compounds) screens alongside KRASG12C inhibition as well as global mass spectrometry based phosphoproteomics (pY/S/T) to examine adaptive signaling changes. KRASG12C mutant lung cancer cell lines have differential sensitivity to KRASG12C inhibition with ARS-1620. Signaling analysis indicates inhibition of p-ERK and to some degree pAKT after 6 hours with evidence of rebound by 24 hr. To expand our understanding of cell type specific response and pathway reactivation upon KRASG12C inhibition, TMT-based quantitative phosphoproteomics was performed after phosphotyrosine (pY) enrichment in 3 cell lines (H358, Calu1 and H1792) after ARS-1620 treatment (6h and 24h). We identified 2275 human phosphosites in total, corresponding to 1153 proteins in 987 protein groups, out of which 433 phosphosites were differentially expressed (1.5-fold change and p-value < 0.05) between any treatment time point and control. Preliminary analysis revealed some common and cell line specific response to ARS-1620. MAPK1, MAPK3, MET, CTNND1, and VTI1B exhibit the strongest down-regulation, across all time points in all cell lines. We observed 84 phosphosites corresponding to 77 proteins that were more abundant in ARS-1620 treated cells indicating a potential for adaptive resistance signaling. Results from the small molecule screens indicate compounds targeting ERBB/HER family of kinases and PI3K pathway showed better synergistic effects with ARS-1620. Signaling analysis in H358, H23 and H2122 cells revealed more abundant tyrosine phosphorylated and total HER2 and HER3 upon ARS-1620 treatment. Further suppression of pERK and pAKT was observed when afatinib was added to ARS-1620 resulting in more combined effects on cell viability. When compared to more specific EGFR inhibition (erlotinib), afatinib enhanced inhibition of pERK and pAKT by ARS-1620 and had more effects on cell viability. However, in H1792 cells, phosphoproteomics identified increased tyrosine phosphorylation of FGFR1 following ARS-1620 and this correlated with combination effects on cell viability of ARS-1620 with the FGFR inhibitor AZD-4547. Lastly, our results suggest that cells sensitive to combination with either HER TKI or FGFR TKI with ARS-1620 are also demonstrating combination effects with SHP2 inhibition (SHP099). Our combined chemical biology and phosphoproteomics results suggest widespread adaptive signaling changes, some of which are contributing to adaptive resistance. Results suggest a more important role of pan-HER inhibition compared with EGFR inhibition. These data may guide biomarker development strategies, especially those that reflect adaptive signaling responses, to predict clinically effective combination strategies in diverse group of KRASG12C mutant lung cancer. Citation Format: Hitendra Singh Solanki, Bin Fang, Eric A. Welsh, Aik C. Tan, Fumi Kinose, Uwe Rix, Eric B. Haura. Adaptive resistance mechanisms to KRASG12C specific inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3410.

Abstract 1889: Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors

Abstract Introduction: Neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) are malignant sarcomas occurring in approximately 15% of NF1 patients and always develop within benign precursor lesions (plexiform neurofibromas). The mortality caused by these tumors is high, approaching 100% in patients with unresectable, metastatic or recurrent disease. Targeted inhibition of the MAP kinase (MAPK) pathway has been attempted in clinical trials using MEK inhibitors (MEKi). While emerging data demonstrate effective tumor growth inhibition in benign plexiform neurofibromas, MEK inhibition blocks MPNST progression only to a limited extent. Combination strategies targeting both the MAPK and PI3K/AKT pathways (horizontal inhibition) have had limited clinical success due to excessive toxicities which precludes delivery of optimal therapeutic concentrations. Thus, novel approaches and complementary treatment strategies that are tolerable and prevent adaptive resistance mechanisms in NF1 MPNST are urgently needed. Methods: We have succeeded in establishing orthotopic in vivo models (patient-derived human xenografts-PDXs and genetically engineered mouse models- GEMMs) via implantation of tumor cells into the sciatic nerve, an approach that is critical, because it preserves the tumor microenvironment. Furthermore, we have optimized MPNST culture conditions, allowing us to establish robust primary cultures of MPNSTs while retaining key biological features of the original tumors. Summary of findings: Using this preclinical testing platform, we demonstrate that - 1. A feedback loop mediated by induction of receptor tyrosine kinase (RTK) signaling becomes hyperactivated upon MEK inhibition (MEKi). This subsequently activates the MAPK pathway to such an extent that MEKi alone is unable to completely block signaling to ERK, thereby maintaining tumor proliferation. 2. SHP2 is a protein tyrosine phosphatase (PTP) that functions as a positive signal transducer, acting between RTKs and RAS and thus, functions as physiological mediator for RAS activation. A treatment paradigm where MEKi is combined with SHP099, an allosteric SHP2 inhibitor to counteract the rebound increase in RTK mediated signaling to RAS-MEK-ERK results in effective attenuation of MEK-ERK signaling. In vivo studies demonstrate that the drug combination exhibits synergistic effects on tumor growth inhibition and significantly prolongs survival. 3. SHP099 disrupts a functional complex (SOS1/GRB2) which is essential to RAS-GTP loading and inhibits RAS-mediated downstream MAP kinase signaling in NF1 MPNST. Conclusions - Our results provide a mechanistic context for SHP2's precise role in the regulation of RAS-GTP and demonstrate that SHP2i can prevent adaptive resistance to MEKi. Given that allosteric SHP2 inhibitors are currently in clinical trials (NCT03114319), our studies can be rapidly translated into a clinical trial to evaluate a combination of SHP2i and MEKi as a novel treatment approach in NF1-MPNSTs. Citation Format: Sameer Farouk Sait, Kwan Tang, Rebecca Brown, Daochun Sun, Xunhua Xie, Benjamin Neel, Luis Parada. Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1889.