Acquired Tyrosine Kinase Inhibitors Resistance Research Articles

BCR::ABL1 Proteolysis-targeting chimeras (PROTACs): The new frontier in the treatment of Ph+ leukemias?

BCR::ABL1 tyrosine kinase inhibitors (TKIs) have turned chronic myeloid leukemia (CML) from a lethal condition into a chronic ailment. With optimal management, the survival of CML patients diagnosed in the chronic phase is approaching that of age-matched controls. However, only one-third of patients can discontinue TKIs and enter a state of functional cure termed treatment-free remission (TFR), while the remainder require life-long TKI therapy to avoid the recurrence of active leukemia. Approximately 10% of patients exhibit primary or acquired TKI resistance and eventually progress to the blast phase. It is thought that recurrence after attempted TFR originates from CML stem cells (LSCs) surviving despite continued suppression of BCR::ABL1 kinase. Although kinase activity is indispensable for induction of overt CML, kinase-independent scaffold functions of BCR::ABL1 are known to contribute to leukemogenesis, raising the intriguing but as yet hypothetical possibility, that degradation of BCR::ABL1 protein may accomplish what TKIs fail to achieve - eliminate residual LSCs to turn functional into real cures. The advent of BCR::ABL1 proteolysis targeting chimeras (PROTACs), heterobifunctional molecules linking a TKI-based warhead to an E3 ligase recruiter, has moved clinical protein degradation into the realm of the possible. Here we examine the molecular rationale as well as pros and cons of degrading BCR::ABL1 protein. We review reported BCR::ABL1 PROTACs, point out limitations of available data and compoundsand suggest directions for future research. Ultimately, clinical testing of a potent and specific BCR::ABL1 degrader will be required to determine the efficacy and tolerability of this approach.

Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC.

Ponatinib and STAT5 Inhibitor Pimozide Combined Synergistic Treatment Applications Potentially Overcome Drug Resistance via Regulating the Cytokine Expressional Network in Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro. The results showed that expression of interleukin (IL) 1B, IL9, and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3, IL8, IL9, IL10, IL12, or IL15. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2, IL6, IL15, IFNG, and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.

Impact of Tumor-intrinsic Molecular Features on Survival and Acquired Tyrosine Kinase Inhibitor Resistance in ALK-positive NSCLC.

In a large-scale, contemporary cohort of patients with advanced ALK-positive NSCLC, we evaluated molecular characteristics and their impact on acquired resistance mutations and clinical outcomes. Our findings that certain ALK variants and co-mutations are associated with differential survival and specific TKI-relevant resistance patterns highlight potential molecular underpinnings of the heterogenous response to ALK TKIs and nominate biomarkers that may inform patient selection for first-line and consolidative therapies.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

Mechanisms of resistance to tyrosine kinase inhibitor treatments in patients with ROS1 fusion-positive non-small cell lung cancer.

9062 Background: ROS1 rearrangement was found in about 1% non-small cell lung cancer (NSCLC), and the fusion proteins created during this process will result in the successive activation of the ROS1 kinase domain. NSCLC patients with ROS1 gene fusions were considered a unique subtype highly sensitive to related tyrosine kinase inhibitor (TKI) treatments. However, the acquired TKI resistance was the major hurdle preventing patients from getting prolonged treatment benefits. Methods: 107 patients diagnosed with advanced or metastatic NSCLC harboring ROS1 fusion were retrospectively recruited. All patients were treated with crizotinib as first-line treatment, and 21 patients received lorlatinib after crizotinib progression. Samples were collected at baseline, after crizotinib progression, and after lorlatinib progression, which all underwent targeted DNA sequencing. TKIs binding to mutated ROS1 fusion proteins was simulated using molecular dynamics simulations. Results: The most witnessed fusion partner of ROS1 was CD74 (58%), followed by SDC4 (14%), EZR (11%), and SLC34A2 (9%). The median progression-free survival was 12.9 months for crizotinib and 6.4 months for lorlatinib. Patients with CD74-ROS1 and SLC34A2-ROS1 had significantly longer PFS than those with other ROS1 fusion types when treated with crizotinib. Patients with baseline TP53 mutations showed worse PFS compared to TP53 wild-type (WT) patients (P < 0.001, HR: 0.19, 95%CI: 0.09-0.39) under crizotinib treatment. An accumulation of both on-target (baseline vs. post-crizotinib vs. post-lorlatinib: 0% vs. 43% vs. 62%) and off-target resistant mutations (baseline vs. post-crizotinib vs. post-lorlatinib: 22% vs. 26% vs. 43%) after multiple TKI treatments was observed. A total of ten on-target resistance mutations were detected after TKI therapies, with 4 first-reported mutations (ROS1 L2010M, G1957A, D1988N, L1982V). According to the molecular dynamics simulation results, ROS1 L2010M mutations (happening in the ROS1 kinase ATP binding cassette) maybe resistant to lorlatinib, entrectinib, cabozantinib, and crizotinib. ROS1 G1957A may lead to resistance to cabozantinib. Moreover, all four novel on-target mutations may lead to resistance to crizotinib. Conclusions: In summary, CD74- and SLC34A2-ROS1 patients showed better crizotinib efficacy with different resistance mutation patterns. Patients of these subtypes are potential beneficiaries of molecular testing after crizotinib progression, directing future treatment strategies. Multiple TKI treatments may lead to the accumulation of both on-target and off-target resistance mutations. In addition, 4 novel ROS1 on-target mutations identified underwent molecular dynamics simulations, unveiling potential resistance to different TKIs, which can provide vital information for future ROS1 fusion-positive NSCLC patient treatment selections.

Integrin αvβ3 Is a Master Regulator of Resistance to TKI-Induced Ferroptosis in HER2-Positive Breast Cancer

Human epidermal growth factor receptor-2 (HER2)-targeting therapies provide clinical benefits for patients with HER2-positive breast cancer. However, the resistance to monotherapies invariably develops and leads to disease relapse and treatment failure. Previous studies have demonstrated a link between the potency of HER2-targeting tyrosine kinase inhibitors (TKIs) and their ability to induce an iron-dependent form of cell death called ferroptosis. The aim of this study was to understand the mechanisms of resistance to TKI-induced ferroptosis and identify novel approaches to overcome treatment resistance. We used mouse and human HER2-positive models of acquired TKI resistance to demonstrate an intimate link between the resistance to TKIs and to ferroptosis and present the first evidence that the cell adhesion receptor αvβ3 integrin is a critical mediator of resistance to TKI-induced ferroptosis. Our findings indicate that αvβ3 integrin-mediated resistance is associated with the re-wiring of the iron/antioxidant metabolism and persistent activation of AKT signalling. Moreover, using gene manipulation approaches and pharmacological inhibitors, we show that this "αvβ3 integrin addiction" can be targeted to reverse TKI resistance. Collectively, these findings provide critical insights into new therapeutic strategies to improve the treatment of advanced HER2-positive breast cancer patients.

CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance.

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) Tcell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

33 (PB023) - Establishment and characterization of a preclinical platform of subcutaneous renal cell carcinoma (RCC) patient-derived xenograft models to evaluate novel treatment strategies

Background: Renal cell carcinoma (RCC) is a cancer type with an onset mainly during the sixth or seventh decade of the patient’s life. Patients with advanced or metastasized RCC have a poor prognosis. The majority of patients develop drug resistance towards Standard of Care (SoC) drugs within months. Tyrosine kinase inhibitors (TKI) are used in first-line therapy and have been combined with immune checkpoint inhibitors recently. The development of novel therapies targeting acquired TKI resistance mechanisms in advanced and metastatic RCC remains an urgent medical need.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

A Molecularly Characterized Preclinical Platform of Subcutaneous Renal Cell Carcinoma (RCC) Patient-Derived Xenograft Models to Evaluate Novel Treatment Strategies.

Renal cell carcinoma (RCC) is a kidney cancer with an onset mainly during the sixth or seventh decade of the patient’s life. Patients with advanced, metastasized RCC have a poor prognosis. The majority of patients develop treatment resistance towards Standard of Care (SoC) drugs within months. Tyrosine kinase inhibitors (TKIs) are the backbone of first-line therapy and have been partnered with an immune checkpoint inhibitor (ICI) recently. Despite the most recent progress, the development of novel therapies targeting acquired TKI resistance mechanisms in advanced and metastatic RCC remains a high medical need. Preclinical models with high translational relevance can significantly support the development of novel personalized therapies. It has been demonstrated that patient-derived xenograft (PDX) models represent an essential tool for the preclinical evaluation of novel targeted therapies and their combinations. In the present project, we established and molecularly characterized a comprehensive panel of subcutaneous RCC PDX models with well-conserved molecular and pathological features over multiple passages. Drug screening towards four SoC drugs targeting the vascular endothelial growth factor (VEGF) and PI3K/mTOR pathway revealed individual and heterogeneous response profiles in those models, very similar to observations in patients. As unique features, our cohort includes PDX models from metastatic disease and multi-tumor regions from one patient, allowing extended studies on intra-tumor heterogeneity (ITH). The PDX models are further used as basis for developing corresponding in vitro cell culture models enabling advanced high-throughput drug screening in a personalized context. PDX models were subjected to next-generation sequencing (NGS). Characterization of cancer-relevant features including driver mutations or cellular processes was performed using mutational and gene expression data in order to identify potential biomarker or treatment targets in RCC. In summary, we report a newly established and molecularly characterized panel of RCC PDX models with high relevance for translational preclinical research.

Abstract 1827: CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance

Abstract Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [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 1827.

Abstract 548: EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC

Abstract The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [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 548.

Abstract P076: Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC

Abstract ROS1 tyrosine kinase inhibitors (TKIs) entrectinib and crizotinib have significantly improved outcomes for patients with ROS1 gene fusion positive (ROS1+) lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately one-third of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in the majority of patients remain unknown or poorly characterized. The purpose of this study was to model and characterize mechanisms of acquired ROS1 TKI resistance. To investigate resistance to ROS1 TKIs, we used patient-derived cell lines CUTO28 (TPM3-ROS1) and CUTO37 (CD74-ROS1) to generate drug-resistant derivatives to entrectinib (CUTO28-ER, CUTO37-ER) or crizotinib (CUTO28-CR,CUTO37-CR) in vitro. We utilized several techniques to probe the mechanisms driving resistance, including DNA and RNA sequencing, fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA sequencing of the ROS1 KD in all resistant cell lines did not reveal any mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in cell proliferation assays. Consistent with MET dependency, MAPK and Akt pathways were inhibited by treatment with a MET-selective, but not a ROS1-selective, TKI. Western blot demonstrated MET overexpression and interphase FISH confirmed MET gene amplification (MET:CEP7 ratio 4.2) compared to parental cells (MET:CEP7 ratio 1.0). Notably, metaphase FISH revealed extrachromosomal DNA (ecDNA) amplification of MET. CUTO28-CR cells displayed sensitivity to Src family kinase (SFK) TKIs, and addition of pan-HER TKI afatinib resulted in synergistic inhibition of proliferation and downstream MAPK signaling. CUTO37-ER cells displayed sensitivity via proliferation and signaling to pan-HER inhibitor afatinib, and phosphorylation of EGFR Y845 consistent with Src-mediated activation of EGFR. Parental CUTO28 and CUTO37 cells were not sensitive to SFK or HER inhibition. Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells—(EGFR:CEP7 ratio 2.1) compared to parental (EGFR:CEP7 ratio 1.0)—but not in CUTO37-ER or CUTO37-CR cells. HER2 gene amplification was not identified in any of the cell lines. Stimulation with epidermal growth factor (EGF) markedly enhanced MAPK signaling in CUTO28-CR, CUTO37-ER, and CUTO37-CR cells to a greater extent than in corresponding parental cells, consistent with EGFR or HER2 dependence. In conclusion, we demonstrated novel mechanisms of resistance to ROS1 TKIs entrectinib and crizotinib. These include ecDNA amplification of MET as an alternate oncogene driver and Src/EGFR axis as a driver of bypass signaling. Combination strategies with existing TKIs could be explored in patients with ROS1 TKI resistance. The discovery of ecDNA MET amplification is particularly intriguing; ecDNA is associated with more aggressive cancers and next generation sequencing analyses do not typically involve amplicon reconstruction to detect ecDNA. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Kristen Turner, Jason Christianson, Robert C. Doebele. Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P076.

In This Issue

In This Issue

The value of defining molecular resistance in patients with progressive EGFR and ALK-driven lung cancer in a public system.

3126 Background: Repeat molecular profiling, except to detect EGFR T790M, is not routinely performed in Canadian patients with lung cancer progressing on EGFR tyrosine kinase inhibitors (TKIs). We performed genomic profiling on post-progression biopsies in patients with stage IV non-small cell lung cancer (NSCLC) and known EGFR/ ALK aberrations treated with TKIs to identify resistance mechanisms, evaluate options for subsequent treatment, and to assess clinical trial eligibility and costs. Methods: From Feb 2018-Aug 2020, post-progression tumour biopsies from consenting patients at a major cancer centre underwent genomic profiling (ThermoFisher OCA v3.0 including hotspots, fusions, and copy number variations in 161 cancer-associated genes). Outcomes of interest were the identification of resistance mutations, actionable targets, clinical trial eligibility (per clinicaltrials.gov), and costs. Results: Thirty-two patients consented to the study. Most, 84% (n = 27), had successful testing completed while 16% (n = 5) had insufficient tissue. Median age of the cohort was 56 yrs, 59% (n = 16) were female, 74% (n = 20) were never-smokers, 81% (n = 22) had ECOG performance status 0-1, and 67% (n = 18) were Asian. The majority, 81% (n = 22) had EGFR mutated NSCLC, and had progressed on EGFR TKIs (15 with previously identified T790M had progressed on osimertinib), and 19% (n = 5) had ALK fusions. Patients had received a median of 2 prior lines of targeted therapy prior to re-biopsy (IQR 1.5,3). One patient had evidence of small cell transformation and associated TP53 and RB1 mutations, 11% (n = 3) had acquired EGFR C797S mutations, and 11% (n = 3) had acquired ALK resistance point mutations (G1202R n = 2, I1171N n = 1). Genomic profiling identified additional actionable targets in 19% of patients (n = 5: MET exon 14 skip mutation n = 1, MET amplification n = 2, BRAF V600E n = 2). Overall, 33% (n = 9) patients had management-changing resistance mechanisms identified (small cell transformation n = 1, actionable targets n = 5, ALK inhibitor resistance = 3). New clinical trial options based on genomic profiling results were identified for 67% (n = 18) of patients. Incremental costs for repeat genomic profiling were approximately $880 CAD per case. Conclusions: Molecular profiling upon development of resistance to targeted therapy in our cohort revealed actionable resistance mechanisms for over a third of patients and clinical trial options for 67%. These incremental benefits for patients highlight the importance of routine molecular profiling in the setting of acquired TKI resistance in lung cancer.

Metabolic Plasticity Is an Essential Requirement of Acquired Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

Simple SummaryTyrosine kinase inhibitors (TKIs), such as imatinib, have become the standard initial treatment of choice for chronic myeloid leukemia (CML) patients. However, one obstacle to face is that a significant proportion of patients presents poor response to TKIs, or acquires resistance resulting in disease relapses. Mutations in BCR-ABL1 protein are a well described mechanism of resistance but other not well established mechanisms outside BCR-ABL1 mutations are emerging as important in the acquisition of resistance. Abnormal metabolism of CML cells that acquire resistance to imatinib has been pointed out as a putative downstream key event, but deep studies aimed to unveil metabolic adaptations associated with acquired resistance are still lacking. Here, we perform an exhaustive study on metabolic reprogramming associated with acquired imatinib resistance and we identify metabolic vulnerabilities of CML imatinib resistant cells that could pave the way for new therapies targeting TKI failure. Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.

Whole-exome sequencing reveals potential mechanisms of drug resistance to FGFR3-TACC3 targeted therapy and subsequent drug selection: towards a personalized medicine

BackgroundDrug resistance is a major obstacle to effective cancer therapy. In order to detect the change in tumor genomic states under drug selection pressure, we use next-generation sequencing technology to investigate the underlying potential mechanisms of drug resistance.MethodsIn our study, we presented a bladder cancer patient who had been a bona fide responder to first-line gemcitabine plus cisplatin regimen and second-line pazopanib (tyrosine kinase inhibitor (TKI) for FGFR3-TACC3 fusion) but finally had disease progression as an ideal case for showing genomic alteration during drug resistance. We applied whole-exome sequencing and ultra-deep target sequencing to the patient pre- and post- pazopanib resistance. Protein-protein interaction (PPI) network and Gene Ontology (GO) analyses were used to analysis protein interactions and genomic alterations. Patient-derived xenograft (PDX) model was built to test drug sensitivity.ResultsTwelve mutations scattered in 12 genes were identified by WES pre- pazopanib resistance, while 63 mutations in 50 genes arose post- pazopanib resistance. PPI network showed proteins from multiple epigenetic regulator families were involved post- pazopanib resistance, including subunits of chromatin remodeler SWI/SNF complex ARID1A/1B and SMARCA4, histone acetylation writers CREBBP, histone methylation writer NSD1 and erasers KDM6A/5A. GO enrichment analysis showed pazopanib resistance genes were prominently tagged for chromatin modification, transcription, as well as gland development, leaving genes with the best adaptive FGFR TKI-coping mechanisms. In addition, significantly elevated tumor mutational burden suggested possible utility of immunotherapy. Intriguingly, PDX model suggested that, sensitivity to original chemotherapy regimen (cisplatin) was restored in patient tumor post-pazopanib.ConclusionsEpigenetic regulation may play a role in acquired TKI resistance. Our study traced the complete tumor genomic variation course from chemo-resistant but TKI-sensitive to TKI-resistant but chemo-(re) sensitive, revealing the potential complex dynamic drug-driven mechanisms of resistance.

Abstract 3003: MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC

Abstract ROS1 gene fusions are predominantly found in non-small cell lung cancer (NSCLC), and ROS1 gene fusion-positive (ROS1+) tumors comprise 1-2% of all diagnosed lung adenocarcinomas. Currently, there are two ROS1 tyrosine kinase inhibitors (TKIs) which are US FDA approved, crizotinib and entrectinib, both of which generate tumor response rates in excess of 70% and demonstrate prolonged disease control. However, acquired resistance to TKIs is inevitable, and has been reported in ROS1+ NSCLC tumors or cancer cell models by our group and others and include ROS1 kinase domain mutations (e.g., L1951R, S1986Y/F, F2004V, L2026M, and G2032R), activation of bypass signaling pathways (e.g., EGFR, RAS, or KIT), or phenotypic transformation (such as epithelial to mesenchymal transition). In order to better understand mechanisms of acquired TKI resistance in ROS1 NSCLC, our lab utilized primary, patient-derived cancer cell models harboring these rare oncogenic kinase rearrangements. Herein, using a patient-derived NSCLC cancer cell line (CUTO28) harboring a TPM3-ROS1 fusion, we derived an entrectinib-resistant cell line (CUTO28-ER) through in vitro culture under drug selection. In these CUTO28-ER cells, we identified: (1) MET-mediated bypass signaling as an acquired resistance mechanism to entrectinib, (2) upregulation of MET signaling is accomplished via MET gene amplification, and (3) resistance could be overcome by the dual ROS1/MET inhibitor crizotinib. To our knowledge, this is the first reported case of MET-mediated acquired resistance in a ROS1+ cancer. This cell line model finding was supported by another primary, patient-derived cell line (CUTO38) harboring a CD74-ROS1 fusion; this cell line was derived from a NSCLC patient following disease progression on entrectenib in the clinic. CUTO38 is resistant to entrectinib in vitro and displays elevated MET expression, MET activation with increased phosphorylation, and dependence on MET for cell proliferation and survival. Notably, MET activation in CUTO38 was not generated by gene amplification, MET exon 14 splicing, MET gene fusion, or autocrine HGF expression, suggesting alternate means of MET activation. These findings suggest that interrogation of MET gene amplification or other MET biomarkers should be performed in ROS1+ NSCLC patients who experience disease progression on entrectinib or other ROS1 TKIs that do not inhibit MET, since there are clinically available MET inhibitors, some of which are dual ROS1/MET inhibitors, that may overcome drug resistance in these patients. Citation Format: Logan Tyler, Anh Le, Hala Nijmeh, Robert Doebele. MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC [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 3003.

Neuropilin-2b facilitates resistance to tyrosine kinase inhibitors in non–small cell lung cancer

ObjectiveInnate and acquired resistance is the principle factor limiting the efficacy of tyrosine kinase inhibitors in lung cancer. We have observed a dramatic upregulation of the cell surface co-receptor neuropilin-2b in lung cancers clinically treated with tyrosine kinase inhibitors correlating with acquired resistance. We hypothesize that neuropilin-2b plays a functional role in acquired tyrosine kinase inhibitor resistance. MethodsNon–small cell lung cancer proliferation and survival were determined during chronic tyrosine kinase inhibitor exposure in the presence or absence of neuropilin-2b knock-down. Interactions of neuropilin-2a and neuropilin-2b isoforms with PTEN and GSK3β were assessed by immunoprecipitation. Neuropilin-2a and neuropilin-2b mutants deleted for their cytoplasmic domains were used to identify regions responsible for neuropilin-2b–GSK3β interaction. Because GSK3β is known to phosphorylate and degrade PTEN, phospho-PTEN and total PTEN levels were assessed after transfection of neuropilin-2a and neuropilin-2b wild-type and mutant constructs. ResultsNon–small cell lung cancer cells chronically treated with gefitinib or osimertinib developed drug resistance and exhibited logarithmic growth in the presence of endothelial growth factor receptor tyrosine kinase inhibitors. However, neuropilin-2b knockdown cells remained sensitive to gefitinib. Likewise, neuropilin-2b knockdown suppressed and neuropilin-2a knockdown enhanced cellular migration. Acquired drug resistance and cell migration correlated with neuropilin-2b-dependent AKT activation with the intermediate step of GSK3β-dependent PTEN degradation. A specific binding site for GSK3β on the cytoplasmic domain of neuropilin-2b was identified with truncated protein constructs and computer modeling. ConclusionsNeuropilin-2b facilitates non–small cell lung cancer resistance to tyrosine kinase inhibitors, and this biological effect relates to AKT activation. Neuropilin-2b GSK3β interactions appear to be essential for PTEN degradation and AKT activation in lung cancer cells. Disruption of the neuropilin-2b GSK3β interaction may represent a novel treatment strategy to preserve sensitivity to tyrosine kinase inhibitors in non–small cell lung cancer.