Adaptive Resistance Mechanisms Research Articles

Antibiofilm Activity of PEGylated Branched Polyethylenimine.

Biofilm formation is an adaptive resistance mechanism that pathogens employ to survive in the presence of antimicrobials. Pseudomonas aeruginosa is an infectious Gram-negative bacterium whose biofilm allows it to withstand antimicrobial attack and threaten human health. Chronic wound healing is often impeded by P. aeruginosa infections and the associated biofilms. Previous findings demonstrate that 600 Da branched polyethylenimine (BPEI) can restore β-lactam potency against P. aeruginosa and disrupt its biofilms. Toxicity concerns of 600 Da BPEI are mitigated by covalent linkage with low-molecular-weight polyethylene glycol (PEG), and, in this study, PEGylated BPEI (PEG350-BPEI) was found exhibit superior antibiofilm activity against P. aeruginosa. The antibiofilm activity of both 600 Da BPEI and its PEG derivative was characterized with fluorescence studies and microscopy imaging. We also describe a variation of the colony biofilm model that was employed to evaluate the biofilm disruption activity of BPEI and PEG-BPEI.

MEK inhibitor resistance in lung adenocarcinoma is associated with addiction to sustained ERK suppression

MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of “drug addiction” in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a “drug holiday” to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.

Non-genetic adaptive resistance to KRASG12C inhibition: EMT is not the only culprit.

Adaptions to therapeutic pressures exerted on cancer cells enable malignant progression of the tumor, culminating in escape from programmed cell death and development of resistant diseases. A common form of cancer adaptation is non-genetic alterations that exploit mechanisms already present in cancer cells and do not require genetic modifications that can also lead to resistance mechanisms. Epithelial-to-mesenchymal transition (EMT) is one of the most prevalent mechanisms of adaptive drug resistance and resulting cancer treatment failure, driven by epigenetic reprogramming and EMT-specific transcription factors. A recent breakthrough in cancer treatment is the development of KRASG12C inhibitors, which herald a new era of therapy by knocking out a unique substitution of an oncogenic driver. However, these highly selective agents targeting KRASG12C, such as FDA-approved sotorasib (AMG510) and adagrasib (MRTX849), inevitably encounter multiple mechanisms of drug resistance. In addition to EMT, cancer cells can hijack or rewire the sophisticated signaling networks that physiologically control cell proliferation, growth, and differentiation to promote malignant cancer cell phenotypes, suggesting that inhibition of multiple interconnected signaling pathways may be required to block tumor progression on KRASG12C inhibitor therapy. Furthermore, the tumor microenvironment (TME) of cancer cells, such as tumor-infiltrating lymphocytes (TILs), contribute significantly to immune escape and tumor progression, suggesting a therapeutic approach that targets not only cancer cells but also the TME. Deciphering and targeting cancer adaptions promises mechanistic insights into tumor pathobiology and improved clinical management of KRASG12C-mutant cancer. This review presents recent advances in non-genetic adaptations leading to resistance to KRASG12C inhibitors, with a focus on oncogenic pathway rewiring, TME, and EMT.

Opposing Effects of KDM6A and JDP2 on Glucocorticoid Sensitivity in T-ALL

Glucocorticoids (GCs) are a cornerstone of front-line T-cell acute lymphoblastic leukemia (T-ALL) treatment. While mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, other mechanisms of adaptive GC resistance are largely unknown. In an unbiased in vivo screen to discover GC resistance mechanisms, we transplanted and treated 10 primary mouse T-ALLs initiated by retroviral insertional mutagenesis with the GC dexamethasone (DEX) (Wandler et al., 2020). GR protein expression was reduced in ~40% of resistant clones overall, including those with Nr3c1 mutations. The remaining 60% of primary relapsed/resistant leukemias are a valuable resource for discovering additional mechanisms of adaptive GC resistance. Using shotgun cloning, we identified new and unique retroviral integrations near Jdp2 with a corresponding increase in expression in different relapsed clones from independent recipient mice transplanted with T-ALL 8633. Jdp2 integrations were not present in any of the other 9 leukemias. Jdp2 was identified as a T-ALL oncogene in previous insertional mutagenesis screens and shown to induce de novo GC resistance in zebrafish thymocytes (Mansour et al., 2018). T-ALL 8633 was the only leukemia in our panel with a Kdm6a mutation, and we hypothesized that Jdp2 and Kdm6a might interact to modulate GC sensitivity. To explore a possible functional interaction between Jdp2 and Kdm6a in GC resistance, we over-expressed JDP2 using lentiviral transduction and inactivated KDM6A using CRISPR/Cas-9 in CCRF-CEM human T-ALL cells. We refer to these cells as EV (empty vector controls), JDP (JDP2-expressing), EV/KDM (KDM6A mutant) or JDP/KDM (JDP2-expressing; KDM6A mutant). The experiment was reproduced in a second independent CCRF-CEM subclone with similar findings. JDP2 over-expression alone conferred minimal DEX resistance in CCRF-CEM cells (Figure 1). By contrast, KDM6A inactivation unexpectedly enhanced GC sensitivity. Interestingly, JDP2 over-expression reversed this sensitivity in one doubly mutant S1 subclone (JDP/KDM1) and induced overt DEX resistance in another (JDP/KDM2). DEX exposure induced cleaved caspase 3 (CC3) expression in CCRF-CEM subclones that correlated with drug sensitivity, with EV/KDM cells showing the highest levels of CC3 and cells expressing JDP2 alone the lowest. Together, these data suggest a model whereby loss-of-function mutations in KDM6A enhance the sensitivity of T-ALL cells to DEX-induced apoptosis, which then results in selective pressure favoring outgrowth of resistant clones with elevated JDP2 expression. We next investigated basal and DEX-induced NR3C1 and GR expression levels. Upon exposure to DEX, EV/KDM cells up-regulated NR3C1 mRNA and GR protein levels to a greater extent than EV controls, whereas JDP cells were less responsive (Figure 2). Furthermore, "double-mutant” JDP/KDM subclones generally behaved similarly to the JDP2 over-expressing cells, supporting the idea that JDP2 can overcome the DEX sensitization conferred by KDM6A inactivation. To address the potential clinical relevance of these data, we analyzed genomic data from 103 pediatric ALL patients (Li et al., 2020). Of 9 patients with T-ALL and available paired transcriptome data from diagnosis and relapse, two harbored KDM6A mutations. One of these leukemias acquired a somatic NR3C1 mutation at relapse and the other uniquely showed a ~10-fold increase in JDP2 mRNA expression. This relapsed T-ALL also had reduced NR3C1 expression as compared to the paired diagnostic sample. Altogether, these data implicate elevated JDP2 expression as a driver of intrinsic and acquired GC resistance in T-ALL by blocking the expected increase in GR expression in lymphoblasts upon GC treatment. Our findings are consistent with other recent studies showing that genetic and epigenetic mechanisms that converge on GR signaling play a central role in ALL relapse (Li et al., 2021; Autry et al., 2020). We also unexpectedly found that KDM6A inactivation sensitizes T-ALL cells to GC treatment and hypothesize that this results in strong selective pressure for the outgrowth of GC-resistant clones due to JDP2 over-expression and other mechanisms. The FDA-approved BCL-2 inhibitor venetoclax and H3K27me3 inhibition are rational therapeutic approaches for potentially overcoming adaptive GC resistance in relapsed patients with KDM6A-mutant T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Deliverability of a Midostaurin Triplet Regimen Incorporating Venetoclax and Low Dose Cytarabine in Non-Adverse Cytogenetic Risk Acute Myeloid Leukaemia: A Sub-Analysis of the Australasian Leukaemia Lymphoma Group (ALLG) Intervene Study

Background Adaptive resistance mechanisms leading to treatment failure in older patients (pts) receiving frontline venetoclax (VEN) combination therapies include emergence/expansion of kinase activating mutations (principally, FLT3-ITD) in pts with non-adverse (NON-ADV) karyotype AML. In both the RATIFY and QuANTUM-First trials, the FLT3 inhibitor was scheduled sequentially after chemotherapy, with no evidence that this approach augmented marrow toxicity. We conducted the Phase 1b/2 study, INTERVENE, to explore the deliverability and preliminary efficacy of a novel triplet regimen incorporating the FLT3/multi-kinase inhibitor midostaurin (MIDO) delivered sequentially after low dose cytarabine (LDC), in tandem combination with VEN. We hypothesized that this triplet schedule would suppress emergence of kinase-activating mutations on VEN-based therapy. MethodsEligibility: Pts with treatment naïve AML (excluding APL), aged ≥60 years and unfit for intensive chemotherapy. Prior hypomethylating agents for antecedent myeloid neoplasms were permitted. Pts with NON-ADV karyotype were enrolled to receive VEN-LDC-MIDO. Treatment (safety run-in): LDC 20 mg/m2 SC D1-10, MIDO 50 mg BD commenced on D11 and continued to D28. After a dose ramp-up in cycle 1 (C1), VEN D1-28 was administered at two dose levels (1= 400 mg, 2= 600 mg). Azole antifungals were prohibited during C1 but allowed from C2 with VEN dose reduced to 100 mg. Molecular studies: NGS using a custom 48-gene Roche KAPA HyperCap panel (sensitivity 1%) and FLT3-ITD PCR-NGS (sensitivity 10-4-10-5) were performed on serial bone marrow samples. RT-qPCR was used to detect NPM1 measurable residual disease (MRD) (sensitivity 10-6). Results First pt enrolled: 7SEP2020. Data cut-off: 31MAY2022. Eighteen pts were enrolled to receive VEN-LDC-MIDO in the run-in phase. Median age was 77 years (range 73-87, 84% age≥75). 44% had secondary AML, including 11% t-AML. 2 pts withdrew consent within the first 7 days due to non-therapy related reasons and were not response evaluable. 30-day mortality was 0%. There were no dose limiting toxicities observed. With a median follow up of 18 months (m), median overall survival (OS) was not reached (95% CI 8.02-NR). Overall response rate (CR+CRi+CRh) by intention-to-treat was 77.8%, with 44.4% CR. Median relapse free survival was 11.7m. To assess treatment deliverability, we evaluated the inter-cycle time for the first 12 cycles among pts achieving CR/CRi/CRh (Fig 1A). The median inter-cycle times ranged from 28-35 days, with majority commencing the next cycle in <42 days. There was no evidence of cumulative lengthening of inter-cycle time with successive treatment cycles, suggesting that MIDO did not promote marrow toxicity when combined with VEN-LDC. Pts received a median number of 9 cycles (range 1-16), with 7 completing ≥12 cycles (Fig 1B). Of these, 3 electively ceased therapy (at C12, C14, C16), with a treatment-free remission (TFR) lasting 3.3-5.7m, from which one relapsed. For pts opting for therapy continuation (TC) >12m (4/7), one has relapsed. Of the remaining 5 (2 TFR, 3 TC) pts with durable remissions, 4 had either NPM1 or IDH2 mutations at enrolment and all achieved undetectable MRD in CR. One additional patient with NPM1 mutationat diagnosis relapsed after C5 with NPM1 negative AML. Molecular data and treatment outcomes are shown in Fig 1B. Two of 16 response-evaluable pts had FLT3 mutations at baseline (1=FLT3-ITD, 1=TKD). Using PCR-NGS to enhance detection of FLT3-ITD microclones, an additional 3 FLT3-ITDs were identified in 2 pts. During VEN-LDC-MIDO, all 4 FLT3-ITDs were suppressed to undetectable levels by PCR-NGS. No pts relapsed with FLT3 variants at the time of progression. Other kinase variants extinguished by VEN-LDC-MIDO include NRAS G12D and KRAS G12D. In contrast, 4 pathways of adaptive resistance were observed at time of treatment failure: emergence of 1) TP53 hotspot variants (N=2), 2) PPM1D frameshift variants (N=2), 3) BAX inactivating variant (N=1), and 4) evolution of KRAS Q61 mutations (N=2), Conclusion After a median follow up of 18m, we report that VEN-LDC-MIDO, with MIDO delivered sequentially after LDC, is tolerable with excellent treatment deliverability. Preliminary efficacy of this triplet is promising, with molecular evidence of FLT3-ITD suppression in all cases so far. A randomized phase 2 comparison of VEN-LDC-MIDO versus VEN-LDC is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity invitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. Invivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

EP08.02-079 The Use of Lung Adenocarcinoma Patient-Derived Xenografts and Organoids to Study GDP-KRAS G12C Inhibitor Resistance

KRAS-mutations are present in 25-30% of the most common form of lung cancer, lung adenocarcinoma (LUAD), and KRASG12C mutations account for 40% of KRAS-mutations in LUAD. Recent development of covalent GDP-KRASG12C inhibitors (G12Ci) have reignited efforts to target the once “undruggable” protein. Preclinical studies have identified mechanisms of adaptive resistance in cell lines. However, patient-derived xenografts (PDX) and xenograft-derived organoids (XDO) have not yet been extensively used to study mechanisms of primary and acquired resistance.

Abstract 1430: N-cadherin is a driver of adaptive radioresistance in malignant brain tumors

Abstract Many subtypes of brain tumors are highly malignant and resistant to chemo- and radio- therapy. Tumor cells can shift their phenotype in response to treatments, the so-called adaptive resistance. Adaptive resistance mechanisms in malignant brain tumors are still poorly understood, and effective treatments have not yet been developed. To unveil such mechanisms, we have developed unique new experimental models to identify the adaptive resistance mechanisms to fractionated radiation in malignant brain tumors. We performed repeated irradiation (2-5Gy every 3-4 days, 3-6 weeks) on 6 human Glioma stem cells (GSCs), 2 mouse GSCs and 4 medulloblastomas (MB) cells in vitro and examined how tumor cells adapt to repeated irradiation. Brain tumor cells demonstrated dynamic adaptation to fractionated irradiation. They rapidly altered cell proliferation, intercellular adhesion, and stemness and acquired strong radioresistance. To identify genes responsible for radio-resistance, we performed RNA-seq analysis and CRISPR library screening using primary and radioresistant cells. We found that N-cadherin was upregulated in the majority of radioresistant GSCs. Stably transfecting N-cadherin in parental GSC rendered them radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which caused an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug-alone controls, supporting clinical translation. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive resistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. These findings also confirmed that our radioresistant models effectively identify new adaptive resistance mechanisms in malignant brain tumors.

Abstract 3242: The role of SERPINE1 in trametinib resistance in low-grade ovarian serous cancer cell line

Abstract Ovarian cancer is the deadliest gynecological cancer. The dismal 5-year survival rate is partly due to late-stage diagnosis. MEK inhibition therapy has been approved by FDA as treatment option for several cancer types. Recently, trametinib has achieved significantly better objective response rate than traditional standard of care therapies in patients with recurrent low-grade ovarian serous cancer (LGOSC). However, adaptive resistance to MEK inhibitors is common and the mechanism is still unclear. To further investigate the mechanism of adaptive resistance, we have established several adaptive trametinib resistant ovarian cancer cell lines. Our result showed that SERPINE1 was significantly induced in a LGOSC cell line, HOC-7, after acquiring trametinib resistance. To determine the role of SERPINE1 in trametinib resistance in LGOSC, overexpression and down-regulation of SERPINE1 in HOC-7 were achieved by transfection with a cDNA clone expressing SERPINE1 and siRNA silencing respectively. The association of SERPINE1 with trametinib resistance was also investigated in a LGOSC mouse model. For in vitro studies, HOC-7 overexpressing SERPINE1 had a 5-fold increase in IC50 to trametinib in comparison to the parental control cell line HOC-7. Furthermore, pERK expression was also up-regulated in HOC-7 overexpressing SERPINE1. By knocking down the expression of SERIPINE1 in trametinib adaptive resistant HOC-7 cells transfected with SERPINE1 siRNA, we found that the cell proliferation was partly reduced in comparison to cells transfected with control siRNA. More importantly, the trametinib adaptive resistant cells became more sensitive to trametinib. For in vivo studies with the LGOSC mouse model, we found that SERPINE1 expression increased after trametinib treatment in LGOSC tumor tissue. Interestingly, the expression of SERPINE1 protein was found to be correlated to ERK phosphorylation in tumor tissues. Furthermore, the measurement of plasma SERPINE1 in LGOSC mouse model and cancer patient revealed that SERPINE1 could be a predictive marker in cancer diagnosis and prognosis. The above observations were further supported by CCLE and TCGA data that SERPINE1 gene expression level correlated with trametinib sensitivity and patient survival. In conclusion, SERPINE1 could facilitate trametinib resistance by ERK phosphorylation, and this protein has the potential to be a therapeutic target, diagnostic and prognostic biomarker for LGOSC. Further analysis by RNA-seq and immunohistochemical staining of trametinib-treated mouse tumor samples will be performed to understand the mechanism of MEK inhibitor adaptive resistance. Citation Format: Chun Wai Ng, Yvonne TM Tsang, David M. Gershenson, Kwong-Kwok Wong. The role of SERPINE1 in trametinib resistance in low-grade ovarian serous cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3242.

Abstract 3911: Proteomic and transcriptomic subtypes of KRASG12C mutant lung cancer

Abstract Sotorasib (AMG510) is the first KRASG12C covalent inhibitor (KRASG12Ci) approved by the FDA in lung cancers harboring oncogenic KRASG12C mutation. However, it remains unclear why some patients show upfront resistance to the KRASG12Ci, and the mechanisms of adaptive resistance. To enhance the efficacy of the KRASG12Ci, several combination regimens are in clinical trials which includes Sotorasib combination with MEK, PD1, SHP2, pan-HER, PD-L1, and EGFR inhibitors. However, it remains unclear how to precisely identify an appropriate cohort for analysis and predict tumor-centric combination strategies that would be effective in the clinic. We hypothesized that subsets of KRASG12C lung cancer could be identified using transcriptomics and proteomics. To test our hypothesis, we performed hierarchical clustering of the microarray dataset on 87 NSCLC KRASG12C tumors and identified three novel transcriptomic subtypes - Subtype 1 had higher expression of extracellular matrix (ECM) genes, which are related to cellular adhesion, and lower expression of EMT genes. Subtype 2 had low ECM expression, high expression in EMT and cell cycle pathway genes suggesting that this is a more invasive subtype. Subtype 2 had lowest expression of small molecule metabolism, suggesting it may be more responsive to KRASG12Ci. Subtype 3 had the lowest expression in interferon gamma/cytotoxic T-cell, T-cell, and B-cell pathways. Subtype 3 had the highest expression of small molecule metabolism, suggesting that it could be more resistant to KRASG12Ci. Having observed these unique clusters, we next hypothesized that mass spectrometry-based expression and phosphoproteomic analysis (pY and pS/T/Y) will further refine these clusters and help identifying new subsets. Our previous work by Stewart et al., demonstrated the importance of proteomics integrated with genomic and transcriptomic analyses to define molecular subtypes in a cohort of 108 squamous cell lung cancer patients. We believe that a similar integrated proteogenomic study is important in context of KRASG12C tumors to understand the signaling diversity and phenotypically categorized them to optimize patient enrichment schemes for effective combination therapy. For that purpose, we performed an integrative proteogenomic analysis of 75 KRASG12C tumors (55 lung, 12 large bowel adenocarcinomas and 8 PDXs) with full clinical follow up and pathology data. We are presently classifying the 75 KRASG12C tumors into phosphoproteomic subtypes. We also carried out targeted exome sequencing for cancer-specific genes. Next, we plan to leverage existing gene expression microarray data and add exome sequencing proteomics, and phosphoproteomics for a comprehensive, protein-centric characterization on these tumors. Importantly, the inclusion of 8 PDXs being co-processed and co-analyzed along with the tumors will enable rapid follow-up experiments to validate the findings into preclinical therapy trials. Citation Format: Hitendra Singh Solanki, Paul A. Stewart, Eric A. Welsh, Victoria Izumi, Bin Fang, Sean J. Yoder, John M. Koomen, Eric B. Haura. Proteomic and transcriptomic subtypes of KRASG12C mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3911.

Abstract 1363: Preclinical development of bispecific CAR-T cells for canine B cell malignancies

Abstract Chimeric antigen receptor (CAR)-T cells targeting CD19 have revolutionized the treatment of B cell malignancies in people. However, post-treatment loss of CD19 has emerged as an adaptive mechanism of resistance. Pet dogs that develop tumors spontaneously in an immune competent setting represent a compelling parallel population of patients to study next generation cancer immunotherapies. Comparative trials at the University of Pennsylvania are investigating the activity of canine CAR-T cells targeting either CD19 or CD20 in canine B cell lymphoma patients and have revealed a favorable safety profile with early signs of anti-tumor activity. Aligned with experiences in human trials, expansion of CD20-negative B cells has been observed in dogs treated with anti-CD20 CAR-T cells. Bispecific CAR-T cells that simultaneously target two antigens to mitigate antigen escape have encouraging efficacy profiles in human patients. Thus, we designed a dual-targeting CAR specific for canine B cell antigens. A second-generation canine CAR construct targeting both CD19 and CD20 with a 28z signaling domain was cloned into the MSGV1 backbone. Chimeric retrovirus encoding RD114 and VSV-G envelope proteins and the CAR construct was made using GP2-293 cells. Jurkat NFAT-GFP reporter cells and primary T cells from dogs diagnosed with high grade B cell lymphoma were retrovirally transduced with bispecific CARs. Antigen specific reactivity was assessed against cell lines with variable CD19 and CD20 expression patterns. The CD19-CD20 CAR was efficiently transduced, with stable expression in both Jurkat and primary canine T cells. Jurkat reporters revealed dual-specific CAR signaling after co-culture with CD19+ and/or CD20+ target cells. Primary canine CAR-T cells expanded in vitro and exhibited CAR-specific proliferation and cytotoxicity against CD19+ CD20+ target cells. Preclinical manufacture of functional, bispecific anti-CD19 anti-CD20 canine CAR-T cells is feasible. A clinical trial recruiting dogs diagnosed with B cell cancer will assess safety and efficacy of this approach in vivo, with the aim of establishing the dog as a compelling model to appraise the ability of bispecific CAR-T cells to overcome therapeutic failures secondary to antigen loss. Citation Format: Matthew J. Atherton, Antonia Rotolo, Nicola J. Mason. Preclinical development of bispecific CAR-T cells for canine B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1363.

Abstract 1125: Elucidating the transcriptomic response to EGFR-targeted therapy in EGFR-driven glioblastoma

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults with a dismal 15-month median survival. Standard therapy consisting of surgical resection, radiation, and temozolomide has been unsuccessful in meaningfully extending survival and preventing recurrence; thus, novel therapeutics are urgently needed. One proposed targeted treatment strategy for GBM involves using small molecule inhibitors against common genetic mutations. Epidermal growth factor receptor (EGFR) is the most commonly overexpressed oncogene in GBM (~56%). While EGFR tyrosine kinase inhibitors (TKI) have shown promise in other cancers, GBM clinical trials with EGFR TKI have failed. One reason for this failure is the development of adaptive therapeutic resistance. Understanding the mechanisms behind drug resistance is essential for the development of novel, effective therapeutics for GBM. To better understand adaptive resistance in GBM, we utilized two genetically engineered mouse astrocyte lines harboring common GBM mutations: Cdkn2a-/-, EGFRvIII (CEv3) and Cdkn2a-/-, Pten-/-, EGFRvIII (CEV3P). CDKN2A and PTEN are commonly deleted or otherwise inactivated tumor suppressor genes in GBM while the vIII variant of EGFR is the single most common oncogene mutation, making it an attractive therapeutic target. Cell lines CEv3 and CEv3P are both sensitive to neratinib, an irreversible second-generation EGFR TKI, at IC50 of 0.24μM and 0.13µM, respectively. To better understand adaptive response to neratinib treatment, we profiled the transcriptome with RNA sequencing at 0, 4, 24, and 48 hours. Our data shows that kinome rewiring is detectable after just 4 hours of treatment and sustained through 48 hours, with differential expression of 70% or more of the expressed kinome. We propose that differentially expressed kinases in response to neratinib can potentially activate alternative signaling pathways that bypass EGFR inhibition, which ultimately confers resistance to EGFR targeted therapy. Furthermore, we hypothesize that the epigenome is directly responsible for this adaptive kinome response through BRD4 dependent enhancer remodeling. Because dual therapy against EGFR and BRD4 has shown promising results in other cancers, targeting the epigenome through BRD4 represents a potential combination therapy with EGFR TKI in GBM. To profile BRD4-associated epigenomic changes, we used Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) to interrogate several regulatory marks (H3K4me1, K3K4me3, H3K27ac) in addition to BRD4. We seek to integrate RNA sequencing and CUT&amp;RUN data to determine if kinases differentially expressed following neratinib treatment correlate with epigenetic marks for their respective enhancer(s). This work will provide insight into the adaptive resistance mechanism of EGFR driven GBM. Citation Format: Benjamin Lin, Julia Ziebro, Kasey R. Skinner, Abigail Shelton, Erin Smithberger, Ryan Bash, Frank B. Furnari, Ryan Miller. Elucidating the transcriptomic response to EGFR-targeted therapy in EGFR-driven glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1125.

Abstract 3275: Bromodomain and extraterminal (BET) protein inhibition sensitize BRAFV600E colorectal cancer to standard targeted therapies

Abstract Background: Standard treatment in BRAFV600E colorectal cancer (CRC) patients has demonstrated an extremely poor prognosis with only little benefits. Interestingly, BRAFV600E CRC has been identified as a subgroup highly epigenetically regulated in its tumorigenesis and maintenance. Our preliminary data demonstrated loss of BET family genes sensitizes BRAFV600E CRC cells to targeted therapies. However, the activity of BET inhibition is unknown in the context of BRAF mutation. Therefore, we investigated the efficacy of BETi combined with standard treatments in BRAFV600E CRC. Methods: CRISPR-based synthetic lethality screen was conducted using RKO, BRAFV600E CRC cell line. Library containing 355 epigenetic genes was introduced and cells were treated with either DMSO, BRAFi (vemurafenib), or BRAFi/EGFRi (cetuximab). Clonal dropouts under control versus BRAFi or BRAFi/EGFRi lead to identifying top candidate genes potentially synthetic lethal with standard treatments. We further compared the response to targeted therapies between individual BET family (BRD2/3/4) knockout and control BRAFV600E CRC cell lines by XTT or clonogenic assays. Furthermore, BRAFV600E CRC PDX models were treated with vehicle, BETi (iBET151), MEKi (binimetinib), or BETi/MEKi doublet. The efficacy of BETi (zen3694) and BRAFi/EGFRi triplet combination is under investigation. Also, we tested a combination of BETi/MEKi or BETi/BRAFi/EGFRi in the BRAFV600E CRC model with acquired resistance to BRAFi/EGFRi. We assessed treatment response by measuring tumors volume changes for 21 days. For BETi/MEKi combination, RNA-seq and RPPA were performed at 7 days to understand the pharmacodynamics of the combination. Results: Our negative selection screen confirmed loss of BRD2, one of the BET family members, sensitizes cells to both targeted treatments (p &amp;lt; 0.05). BRD2 or BRD4 KO CRC cell lines have shown growth inhibition with BRAFi or BRAFi/EGFRi treatments compared to control (p &amp;lt;0.05). Intriguingly, the efficacy study showed selective tumor regression under the combination of BETi/MEKi (-39% volume change in best responding model) while the effect of individual treatment was limited. We also identified potential adaptive resistance mechanisms to BETi treatment, associated with upregulation of key oncogenic signaling and cell cycle regulators, such as MAPK signaling, MYC, or E2F targets; which were significantly inhibited when combined with MEKi. Lastly, we found stabilized tumor growth induced by both combinations of BETi/MEKi and BETi/BRAFi/EGFRi in the resistant PDX model compared to targeted treatment alone (p &amp;lt;0.05). Conclusion: Our data showed combination of BET inhibition improved sensitivity or overcame resistance to standard targeted therapy in BRAFV600E CRC. Overall, our data suggest that combining BETi with MAPK inhibition could be a novel therapeutic solution for BRAFV600E CRC. Citation Format: Hey Min Lee, Stefania Napolitano, Alexy Sorokin, Melanie N. Woods, Saikat Chowdhury, Jumanah Y. Alshenaifi, Anand K. Singh, John Paul Shen, Funda Meric-Bernstam, Kunal Rai, Scott Kopetz. Bromodomain and extraterminal (BET) protein inhibition sensitize BRAFV600E colorectal cancer to standard targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3275.

Abstract LB193: Chemical genomics identify novel druggable nodes and resistance pathways in the presence of concomitant SOS1 and KRAS inhibition

Abstract KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the MAPK pathway. Recent data has consistently demonstrated co-dependencies of mutant-KRAS with extrinsic proteins that augment GTP-loading. Son of Sevenless homolog 1 (SOS1) is the most proximal of these proteins to KRAS and functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases, thus representing a highly sought-after druggable target. Utilizing a structure-based drug discovery approach, we identified a selective and potent SOS1 inhibitor, MRTX0902, that functions by disrupting the KRAS/SOS1 protein-protein interaction, ultimately preventing SOS1-mediated nucleotide exchange on KRAS. MRTX0902 enhances the anti-tumor activity of the KRASG12C inhibitor adagrasib across multiple KRASG12C-mutant preclinical models; however, it is anticipated that deeper pharmacological inhibition of KRAS signaling may give rise to novel combination targets and additional adaptive resistance mechanisms. To identify additional vulnerabilities and combination strategies, we conducted drug-anchored CRISPR screens in two KRASG12C xenograft models in the presence of MRTX0902 and adagrasib in vitro and in vivo. Interestingly, we found that the SOS1 homolog, SOS2, only emerges as a co-dependency under selective pressure of combination treatment and likely compensates for the inhibition of SOS1. Furthermore, multiple components from the PIK3CA/mTOR signaling pathway, PRMT5, and other non-vertical signal transduction pathways were uncovered and identified as genetic co-dependencies that emerged under selective pressure. Conversely, tumor suppressor genes including TSC1/2, PTEN, NF1, KEAP1 and TP53 along with phosphatases PTPN12 and PTPN14 enhanced tumor growth when knocked out providing a catalogue of putative resistance genes and mechanisms. Lastly, we utilized small molecule inhibitors of putative therapeutic targets identified from our CRISPR screens to validate genetic co-dependencies across in vitro and in vivo translational models. These studies uncover the potential utility of additional drug partners for the MRTX0902 and adagrasib combination and aide in the understanding of SOS and RAS biology in targeted cancer therapy. Citation Format: Shilpi Khare, Niranjan Sudhakar, David M. Briere, Larry Yan, Lars D. Engstrom, Jade Laguer, James Medwid, Laura Vegar, Darin Vanderpool, Matthew A. Marx, John M. Ketcham, James G. Christensen, Peter Olson, Jacob R. Haling. Chemical genomics identify novel druggable nodes and resistance pathways in the presence of concomitant SOS1 and KRAS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB193.

Engineering Bioinspired Nanomedicines to Mitigate the Resistance to Cancer Immunotherapy

ConspectusThe current era has witnessed the success of immunotherapy, in particular, immune checkpoint blockade (ICB) therapy, at an unprecedented pace. However, immunotherapy often fails to unleash the antitumor immune response because of the paucity of appropriate therapeutic targets in the complex tumor microenvironment (TME) and the occurrence of intrinsic and adaptive immune resistance of tumor cells. In recent years, we have rationally engineered a set of bioinspired stimuli-activatable nanotherapeutics to circumvent cancer immune resistance and potentiate cancer immunotherapy. To prompt the development of nanomedicine-based cancer immunotherapy, in this Account we first introduced the mechanisms of intrinsic and adaptive immune resistance from a tumor cell perspective. We then summarized the bioinspired nanomedicine strategies exploited in our laboratory in the past few years to circumvent both the intrinsic and adaptive immune resistance. These nanoparticles were rationally engineered for highly tumor-specific drug delivery of the immunotherapeutics by responding to the endogenous signals of TME including extracellular/intracellular acidity, overexpressed enzymes, glutathione, and reactive species. These nanoparticles caused the intrinsic immune resistance to migrate by restoring tumor-associated antigen presentation on the surface of tumor cells, repolarizing the tumor-associated macrophages, and eliciting antitumor immunogenicity via the induction of the immunogenetic cell death of tumor cells. Meanwhile, these nanoparticles were also loaded with small molecular or macromolecular immune inhibitors to relieve the inducible expression of the immune checkpoints (e.g., programmed cell death of ligand 1 and indolamine 2,3-dioxygenase 1), thereby relieving the adaptive immune resistance. Overviews of the nanomedicine strategies used to cause the immune resistance to migrate by regulating lactate metabolism and lipid peroxidation were also provided. Furthermore, we suggested rational combination strategies according to diverse cancers to mitigate multiple immune resistance. These tactics can be adopted to perpetuate the cancer immunity cycle for clinical therapy of immune-resistant cancer. It is envisaged that TME-activatable bioinspired nanomedicines to bypass immunosuppressive signaling pathways may broaden the clinical impact of cancer immunotherapy.

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET–rearranged thyroid cancer.

Dual targeting of FGFR3 and ERBB3 enhances the efficacy of FGFR inhibitors in FGFR3 fusion-driven bladder cancer

BackgroundMutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents.MethodsAcquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis.ResultsLong term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation.ConclusionsWe demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

Adaptive antimicrobial resistance, a description of microbial variants, and their relevance to periprosthetic joint infection.

Periprosthetic joint infection (PJI) is a difficult complication requiring a comprehensive eradication protocol. Cure rates have essentially stalled in the last two decades, using methods of antimicrobial cement joint spacers and parenteral antimicrobial agents. Functional spacers with higher-dose antimicrobial-loaded cement and antimicrobial-loaded calcium sulphate beads have emphasized local antimicrobial delivery on the premise that high-dose local antimicrobial delivery will enhance eradication. However, with increasing antimicrobial pressures, microbiota have responded with adaptive mechanisms beyond traditional antimicrobial resistance genes. In this review we describe adaptive resistance mechanisms that are relevant to the treatment of PJI. Some mechanisms are well known, but others are new. The objective of this review is to inform clinicians of the known adaptive resistance mechanisms of microbes relevant to PJI. We also discuss the implications of these adaptive mechanisms in the future treatment of PJI. Cite this article: Bone Joint J2022;104-B(5):575-580.

Using Structure-guided Fragment-Based Drug Discovery to Target Pseudomonas aeruginosa Infections in Cystic Fibrosis.

Cystic fibrosis (CF) is progressive genetic disease that predisposes lungs and other organs to multiple long-lasting microbial infections. Pseudomonas aeruginosa is the most prevalent and deadly pathogen among these microbes. Lung function of CF patients worsens following chronic infections with P. aeruginosa and is associated with increased mortality and morbidity. Emergence of multidrug-resistant, extensively drug-resistant and pandrug-resistant strains of P. aeruginosa due to intrinsic and adaptive antibiotic resistance mechanisms has failed the current anti-pseudomonal antibiotics. Hence new antibacterials are urgently needed to treat P. aeruginosa infections. Structure-guided fragment-based drug discovery (FBDD) is a powerful approach in the field of drug development that has succeeded in delivering six FDA approved drugs over the past 20 years targeting a variety of biological molecules. However, FBDD has not been widely used in the development of anti-pseudomonal molecules. In this review, we first give a brief overview of our structure-guided FBDD pipeline and then give a detailed account of FBDD campaigns to combat P. aeruginosa infections by developing small molecules having either bactericidal or anti-virulence properties. We conclude with a brief overview of the FBDD efforts in our lab at the University of Cambridge towards targeting P. aeruginosa infections.

The analysis of the biochemical diversity of the English oak vegetative sphere in the forest-steppe zone

The paper studies the biochemical diversity of the English oak trees growing in the protective forest strips of the forest-steppe zone in the Central Chernozem region. The features of accumulation of various groups of substances of secondary metabolism of phenolic nature in the leaves of model oak trees in seasonal dynamics are described. The role of secondary metabolites in the formation of adaptive mechanisms of resistance of individuals to adverse environmental factors is considered. It is proved that the content of substances of secondary metabolism of phenolic nature and their combination can serve as a criterion for the potential biochemical stability of trees when growing in unfavorable environmental conditions. The content of secondary metabolism substances was determined on oak leaf samples in early June and August. It was revealed that the quantitatively leading group of substances of secondary metabolism are flavonols. It was found that at the beginning of the growing season, the most significant fluctuations in the levels of accumulation of secondary metabolism substances occur in young oak leaf blades, and by the end of the growing season their content stabilizes. High rates of variation coefficients have been identified, which make it possible to ensure biochemical diversity in the population and a sufficient range of adaptability of traits. It is shown that the samples of control and experimental trees for flavonols are the most homogeneous.