RTK Activation Research Articles

Chapter 13: What is new in fibroblastic/myofibroblastic tumors in children.

Fibroblastic and myofibroblastic neoplasms represent about 12% of pediatric soft tissue tumors. Most of these neoplasms in children are either benign or locally aggressive with rare metastasis, while malignant cases are uncommon. Diagnosing these tumors is challenging due to overlapping morphologies and the limited utility of immunohistochemistry. Advances in molecular techniques, especially RNA sequencing, have improved our understanding of the molecular drivers of these tumors, leading to better classification. Key molecular alterations, such as RTK and MAPK activation, are central in the development of tumors like infantile fibrosarcoma (IFS) and inflammatory myofibroblastic tumors (IMT). The identification of alternative fusions in IFS and IMT underscores the importance of an integrated diagnostic approach. Furthermore, new RTK-driven lesions, now included in the WHO's "NTRK-rearranged mesenchymal neoplasms", have been identified. This review provides an update on recent findings in RTK-driven myofibroblastic tumors and highlights novel entities still in need of classification.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAFV600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation – features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of “minority mitochondrial outer membrane permeabilization (MOMP)” – a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in mutant BRAF PTC.

Abstract 1924: RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical models

Abstract Background: KRASG12C-GDP Inhibitors such as sotorasib and adagrasib have demonstrated clinical benefit in lung cancer patients harboring an oncogenic KRASG12C mutation. However, the durability of monotherapy benefit is limited by the development of resistance. Hence, identification of novel treatment approaches to overcome resistance and extend duration of benefit becomes necessary to address this unmet clinical need. Experimental Design: We generated two NSCLC models of KRASG12C−GDP inhibitor acquired resistance, named sotorasib-R LU65 (cell-line derived, in vitro) and adagrasib-R LUN156 (PDX-derived, in vivo) following extended treatment with sotorasib and adagrasib, respectively. We then performed transcriptomic and mass spectrometry-based phosphoproteomics analyses on baseline and KRASG12C-GDP inhibitor-resistant samples to determine resistance and escape mechanisms. Results: In the sotorasib-R LU65 cells, we discovered upregulation of various RTKs including HER2, HER3, and AXL using a mass spectrometry-based proteomics approach and Immunoblot analysis. Simultaneously, from a transcriptome analysis, we observed activation of RAS and PI3K/AKT signaling in these cells. In the adagrasib-R LUN156 model, increased phosphorylation of MET was observed via phospho-RTK array and by immunoblotting. Transcriptomic analysis revealed an enrichment for RAS pathway dependency in the adagrasib-R tumor models. RAS GTP-binding assessed by RAF-RBD pulldown and multiple reactions monitoring mass-spectrometry suggest increased GTP-bound RAS isoforms in sotorasib-R LU65 cells and active RAS signaling in adagrasib-R LUN156. Given the convergence of our findings on re-activation/maintenance of wild-type RAS signaling as a means of resistance to KRASG12C-GDP-state inhibitors, we hypothesized that RMC-7977, a tri-complex RASMULTI-GTP inhibitor, which inhibits signaling via mutant and wild-type RAS, had the potential to counteract these mechanisms. We tested RMC-7977 monotherapy, which drove complete tumor regressions in the adagrasib-R LUN156 model. Additionally, RMC-7977 attenuated tumor growth in sotorasib-R cell derived LU65 xenograft model wherein we observed co-activation of PI3K/AKT signaling. Thus, we tested a combination with the pan-PI3K inhibitor pictilisib and observed combinatorial activity consistent with our findings. Conclusions: These data suggest that in KRASG12C-GDP inhibitor-resistant models RTK activation maintains MAPK dependency and in the case of one model, PI3K signaling. Consistent with these mechanisms, RMC-7977 as a single agent or in combination with pictilisib drove significant tumor regressions in these models. These preclinical results indicate RASMULTI-GTP inhibition alone or combination with PI3K inhibition has the potential to address KRASG12C-GDP inhibitor resistance. Citation Format: Hitendra S. Solanki, Harshit Shah, Denis Imbody, Bina Desai, Paul A. Stewart, Bin Fang, Yaakov Stern, Lancia N. Darville, John M. Koomen, Andriy Marusyk, Ethan Ahler, Ida Aronchik, Mallika Singh, Eric B. Haura. RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1924.

Transcriptomic and genomic profiling of multiple primary colorectal cancers reveals intratumor heterogeneity and a distinct immune microenvironment

This study reported on the intratumor genomic and immunological heterogeneity of different tumor lesions from a single patient with multiple primary colorectal cancer (MPCC). The goal of this study was to explore the molecular and microenvironment characteristics of tumor lesions from different primary sites in a patient with MPCC.A total of three tumor lesions located in the hepatic flexure of the transverse colon, sigmoid colon, and rectum were collected from a 72-year-old male patient with MPCC. All three tumor samples were examined by using whole-exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq). The transcriptome data of The Cancer Genome Atlas (TCGA) colon cancer (COAD) dataset were explored to characterize the biological impacts of certain immune cells. Only three nonsynonymous mutations were shared by all of the tumor lesions, whereas a number of single nucleotide variant (SNV) and copy number variation (CNV) mutations were shared by tumor samples from the sigmoid colon and rectum. Transcriptomic analysis showed that tumor lesions derived from the transverse colon had decreased levels of RTK, ERK, and AKT pathway activity, thus suggesting lower oncogenic properties in the transverse lesion compared to the other two samples. Further immune landscape evaluation by using single-cell transcriptomic analysis displayed significant intratumor heterogeneity in MPCC. Specifically, more abundant mucosal-associated invariant T (MAIT) cell infiltration was found in transverse colon tumor lesions. Afterwards, we found that higher MAIT cell infiltration may correlate with a better prognosis of patients with colon cancer (immunohistochemical status was MSI-L/pMMR) by using a publicly available TCGA dataset.

Design and synthesis of certain 7-Aryl-2-Methyl-3-Substituted Pyrazolo{1,5-a}Pyrimidines as multikinase inhibitors

Four new series 7a-e, 8a-e, 9a-e, and 10a-e of 7-aryl-3-substituted pyrazolo[1,5-a]pyrimidines were synthesized and tested for their RTK and STK inhibitory activity. Compound 7d demonstrated potent enzymatic inhibitory activity against TrkA and ALK2 with IC50 0.087and 0.105 μM, respectively, and potent antiproliferative activity against KM12 and EKVX cell lines with IC50 0.82 and 4.13 μM, respectively. Compound 10e showed good enzyme inhibitory activity against TrkA, ALK2, c-KIT, EGFR, PIM1, CK2α, CHK1, and CDK2 in submicromolar values. Additionally 10e revealed antiproliferative activity against MCF7, HCT116 and EKVX with IC50 3.36, 1.40 and 3.49 μM, respectively; with good safety profile. Moreover, 10e showed cell cycle arrest at the G1/S phase and G1 phase in MCF7 and HCT116 cells with good apoptotic effect. Molecular docking studies were fulfilled for compound 10e and illustrated good interaction with the hot spots of the active site of the tested enzymes.

The mystery of titan hunter: Rationalized striking of the MAPK pathway via Newly synthesized 6‐Indolylpyridone‐3‐Carbonitrile derivatives

MAPK pathway sparkles with RTK activation, passes through subsequent downstream RAS-RAF-MEK-ERK signaling cascades, with consequent direct and indirect CDK4/6 signaling activation, and ends with cell survival, division, and proliferation. However, the emergence of anomalies such as mutations or overexpression in one or more points of the pathway could lead to cancer development and drug resistance. Therefore, designing small inhibitors to strike multitudinous MAPK pathway steps could be a promising synergistic strategy to confine cancer. In this study, twelve 6‐indolylpyridone‐3‐carbonitrile candidates were synthesized and assessed in vitro for antineoplastic activity using four cancer cell lines. The initial antiproliferative screening revealed that compounds 3g, 3h, and 3i were the most potent candidates (GI% Avg = 70.10, 73.94, 74.33%, respectively) compared to staurosporine (GI% Avg = 70.99%). The subsequent safety and selectivity assessment showed that 3h exhibited sub-micromolar inhibition against lung cancer cells (HOP-92 GI50 = 0.75 μM) and 13.7 times selectivity toward cancerous cells over normal cells. As a result, 3h was nominated for deep mechanistic studies which evidenced that compound 3h impressively blocks multiple keystones of the MAPK pathway with nanomolar potency (EGFRWT IC50 = 281 nM, c-MET IC50 = 205 nM, B-RAFWT IC50 = 112 nM, and CDK4/6 IC50 = 95 and 184 nM, respectively). Surprisingly, 3h showed a remarkable potency against mutated EGFR and B-RAF, being 4 and 1.3 more selective to the mutated enzymes over the wild-type forms (EGFRT790M IC50 = 69 nM and B-RAFV600E IC50 = 83 nM). Ultimately, combined molecular docking and molecular dynamics (MD) calculations were executed to inspect the mode of binding and the complex stability of 3h towards the keystones of the MAPK pathway.

Abstract B015: Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib

Abstract Purpose: Sotorasib (AMG510) has demonstrated remarkable response in lung cancer patients with tumors driven by oncogenic KRASG12C mutation. However, recently published clinical data identified acquired mutations in RAS and other genomic alterations as potential mechanisms of resistance. The cause of resistance in more than half of the patient cohort was undetermined with genomic sequencing. We hypothesize that rewiring of signaling networks could be the newly acquired vulnerabilities in cells progressing after sotorasib treatment. Experimental Design: We performed whole exome sequencing, transcriptomic profiling, and mass spectrometry-based proteomics/phosphoproteomics of the parental and sotorasib resistant isogenic line of LU65 (LU65-AMGR). The omics analyses were integrated with functional screens to prioritize a set of druggable targets/pathways. Results: We did not observe obvious acquired mutations that drive resistance to sotorasib in LU65-AMGR cells. To investigate compensatory signaling critically important for the survival/growth of LU65-AMGR cells, we studied signaling perturbations using mass spectrometry-based phosphoproteomics approach. We identified 430 and 1,574 phosphosites differentially expressed in resistant cells (± 1.5-fold & p < 0.05) with phosphotyrosine (pY) and global phosphoproteome (pS/T/Y) enrichment, respectively. Analysis of phosphoproteomics data identified increased phosphorylation of multiple RTKs including EGFR, HER2, HER3, IGF1R, AXL and PDGFRA, including others. RAS-GTP pull-down show increased levels of RAS-GTP and NRAS-GTP in the resistant cells. RAS isoform quantification using parallel reaction monitoring (MRM) mass spectrometry further confirmed increased activity of NRAS in pull-down samples. Transcriptomic analysis revealed elevated genes responsible for anti-apoptosis pathways mediated by PI3K/AKT signaling. Enrichment of transcription factors such as AP1 and AP-2A could drive enhanced EGF expression and EGFR phosphorylation in sotorasib resistant cells. Consistent with enhanced RTK and RAS activity, Western blots confirmed higher phosphorylation of ERK and AKT in LU65-AMGR cells. We show higher EGFR phosphorylation in resistant cells when compared to the parental counterpart. While LU65 cells viability is reduced markedly by sotorasib combination with afatinib, an irreversible HER family inhibitor, resistant cells showed lesser effect on cell viability with afatinib combination. Consistent with our in vitro observations, the sotorasib and afatinib combination treatment significantly regressed tumor growth only in LU65 xenografts, but not in LU65-AMGR xenografts. Conclusions: Our data suggest that activation of multiple RTKs maintains RAS activity and PI3K signaling in LU65-AMGR cells. Thus, dual pan-RAS and PI3K inhibition could serve as promising strategy of treatment in relapsed tumors identified with WT RAS and PI3K signaling activation. Citation Format: Denis Imbody, Hitendra S. Solanki, Bina Desai, Paul A. Stewart, Yaakov Stern, Ryoji Kato, Anurima Majumder, Liznair Bridenstine, Aobuli Xieraili, Bin Fang, Lancia Darville, Fumi Kinose, John M. Koomen, Uwe Rix, Andriy Marusyk, Eric B. Haura. Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B015.

Abstract 5487: Synergy between FAK and PI3K inhibitors in cervical and pancreatic cancer cells

Abstract The Ras/PI3K/ERK signaling network is frequently mutated in many cancer types including cervical cancer and pancreatic cancer. Our previous study suggested that PI3K and FAK form a positive feedback loop to modulate ERK activation. We tested combined inhibition of FAK and PI3K on the growth of cervical and pancreatic cancer in vitro. We found that FAK and PI3K inhibitors synergistically inhibited the growth of some cervical cancer and pancreatic cancer cell lines. The synergy was due to both increased apoptosis and decreased cell proliferation. Molecularly, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors induced activation of several RTK including insulin R and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Together, our results show the promise of combining FAK and PI3K inhibition for cervical cancer and pancreatic cancer treatment, but biomarkers for sensitivity to the combination are needed, and concomitant RTK inhibition may be required to combat potential resistance to the combination. Citation Format: Chao-Cheng Chen, Suyang Wang, Jr-Ming Yang, Chuan-Hsiang Huang. Synergy between FAK and PI3K inhibitors in cervical and pancreatic cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5487.

Abstract 424: RTK inhibitor resistance in NSCLC harboring MED12 mutation is overcome by only blocking MEK signaling, not AKT due to mutated MED12-induced YAP-PTEN dysregulation

Abstract The advent of receptor tyrosine kinase inhibitors (RTKi) are important contributions to treat NSCLC patients harboring genetic aberration effectively. Nevertheless, the emergence of acquired or intrinsic RTKi resistance by other RTK activation bypass finally limits their efficacy. First of all, in this study, we identified MED12 mutation in TKI-resistant NSCLC cells based-on NGS analysis. Although MED12 was known to controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling reportedly, we additionally found that MED12 mutation activates not only TGF-beta receptor not also other ones, such as EGFR, meaning that blocking TGF-beta receptor or EGFR alone may not effective to overcome MED12 mutation-induced RTKi resistance. To overcome the RTKi resistance in NSCLC harboring MED12 mutation, we elaborated the two downstream signaling of RTK in MED12 knock-out NSCLC cells and observed that PI3K/AKT signaling is downregulated but MEK/ERK signaling is upregulated. We observed that only MEK inhibitor (MEKi), not PI3K/mTOR inhibitor, shows effective anti-cancer effect. Therefore, we explored why MEKi alone is effective in RTKi resistant NSCLC cells harboring MED12 mutation and presented that MED12 mutation directly suppressed activation of YAP via blocking a large mediator complex of MED12, MED13, CDK8 and CCNC, thereby increasing PTEN expression by inhibition of miR-29 expression. Increased PTEN inhibits reactivation of PI3K/AKT pathway. In conclusion, we first identified that MED12 mutation induces RTKi resistance though activating other RTKs, such as TGF-beta receptor or EGFR, and blocks PI3K/AKT pathway via dysregulation of YAP/PTEN/AKT interaction. Therefore, among two downstream signaling of RTK, activated MEK/ERK pathway can be a definitive target enough to overcome RTKi resistant NSCLC patients harboring MED12 mutation and MEKi could be a primary treatment option for resistant patients harboring MED12 mutation among NSCLC patients with repetitive recurrence of RTKi resistance by another RTK activation bypass. Citation Format: Hyun-Min Ryu, Deokhoon Kim, Shinkyo Yoon, Ho-Su Lee, Eunjin Lee, Chang Hoon Lee, Wanlim Kim, Seyoung Seo, Kyung Hae Jung, Sook Ryun Park, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. RTK inhibitor resistance in NSCLC harboring MED12 mutation is overcome by only blocking MEK signaling, not AKT due to mutated MED12-induced YAP-PTEN dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 424.

312 (PB092) - Mechanisms of resistance toward FGFR4 inhibition in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC), which accounts for up to 90% of all primary liver cancers, is a leading cause of cancer mortality worldwide. FGF19 is a gut secreted endocrine hormone that acts in liver through its unique receptor FGFR4 and co-receptor KLB, to regulate bile acid synthesis and hepatocyte proliferation. FGF19 is reported to be overexpressed and/or amplified in ∼30% of HCC. Genomic and functional studies have identified FGF19 as a potential driver oncogene in HCC. Several FGFR4 inhibitors have entered clinical trials for treating HCC patients. BLU-554 is a potent, selective, and irreversible FGFR4 inhibitor that has demonstrated preliminary efficacy as a second-line treatment in HCC patients harboring FGF19 overexpression. The observed responses, however, on average only last few months before patient relapse, suggesting likely occurrence of acquired resistance. Here, we investigated the changes that occur with long-term FGFR4 inhibition and the development of resistance in preclinical HCC models, in order to identify resistance mechanisms and possible combination strategies. Materials and methods: We established resistant cell lines following long-term exposure of Huh7 with BLU-554. Proteomic, genomic and transcriptomic characterizations were performed to identify potential resistance mechanisms. Results: Huh7 cells resistant to BLU-554 were generated and confirmed with insensitivity to BLU-554. In addition, the cells were also resistant to other FGFR4 inhibitors including FGF-401, H3B-6527 and erdafitinib. Genomic analysis found no mutations on the FGFR4 gene itself. Proteomic analysis revealed activation of a RTK in the BLU-554 resistant cells compared with the parental cells. Transcriptomic analysis revealed changes in gene expression and GSEA analysis revealed multiple relevant gene sets were significantly enriched in the resistant cells. We tested several inhibitors against the identified RTK pathway and found complete inhibition this pathway could restore BLU-554 sensitivity. Conclusions: These data suggest the bypass activation of other RTK may contribute to acquired resistance of FGFR4 inhibition and potential combination strategy to overcome resistance. No conflict of interest.

Nanoscale Mapping of EGFR and c-MET Protein Environments on Lung Cancer Cell Surfaces via Therapeutic Antibody Photocatalyst Conjugates.

Receptor tyrosine kinases are involved in essential signaling roles that impact cell growth, differentiation, and proliferation. The overexpression or mutation of these proteins can lead to aberrant signaling that has been directly linked to a number of diseases including cancer cell formation and progression. This has led to intense clinical focus on modulating RTK activity through direct targeting of signaling activity or cell types harboring aberrant RTK behavior. In particular, epidermal growth factor receptor (EGFR) has attracted intense clinical attention due to the impact of inhibiting this RTK on tumor growth. However, mutations incurred through targeting EGFR have led to therapeutic resistance that involves not only direct mutations to the EGFR protein but also the involvement of other RTKs, such as c-MET, that can overcome therapeutic-based EGFR inhibition effects. This has, not surprisingly, led to co-targeting strategies of RTKs such as EGFR and c-MET to overcome resistance mechanisms. While the ability to co-target these proteins has led to success in the clinic, a more comprehensive understanding of their proximal environments, particularly in the context of therapeutic modalities, could further enhance both our understanding of their signaling biology and provide additional avenues for targeting these surface proteins. Thus, to investigate EGFR and c-MET protein microenvironments, we utilized our recently developed iridium photocatalyst-based microenvironment mapping technology to catalog EGFR and c-MET surface environments on non-small cell lung cancer cell lines. Through this approach, we enriched EGFR and c-MET from the cell surface and identified known EGFR and c-MET associators as well as previously unidentified proximal proteins.

Abstract 3329: Efficacy of EGFR/PI3K signaling inhibition is enhanced with LSD1 inhibition in glioblastoma stem cell (GSC) models

Abstract Mutated and dysregulated protein kinases, such as EGFR and PI3K, have become major targets in cancer therapy due to the growth advantage they confer and the frequency of alterations. In glioblastoma (GBM), EGFR and PI3K are two of the most mutated genes and result in hyperactivation of these kinases. However, single agent inhibition has offered minimal success, largely due to the development of resistance from compensatory downstream signaling pathways. One strategy to circumvent resistance and improve efficacy in GBM is to use combination therapy, such as concurrent inhibition of EGFR or PI3K with inhibition of relevant epigenetic modulators. In GBM, lysine-specific demethylase 1 (LSD1) is an important epigenetic regulator that has shown to promote kinase signaling in cancer models. Therefore, the present study sought to define the relationship between the EGFR/PI3K signaling pathway and LSD1 and to evaluate the efficacy of co-inhibition of EGFR/PI3K and LSD1 in GBM.Transcriptional changes were examined following knockdown of LSD1 in isogenic human GBM cells using RNA-seq. These data were analyzed by GSEA to evaluate biological processes associated with the LSD1 expression. We identified several kinase signaling processes that were enriched in GBM cells with wild type LSD1 such as gene sets for regulation of PI3K activity, RTK binding, and transmembrane RTK activity. The effect of kinase inhibition on LSD1 expression was assessed using western blot analysis. We also evaluated the effects of LSD1 inhibition on expression of downstream kinase signaling proteins. Three kinase inhibitors directed against either EGFR or PI3K were evaluated in GSCs as single agents and in combination with LSD1 inhibitors. The three kinase inhibitors, osimertinib, erlotinib, and BKM120, all have evidence of some brain penetrance. We also evaluated a novel dual kinase inhibitor, MTX-241, which targets both EGFR and PI3K simultaneously. Five LSD1 inhibitors were assessed for their ability to enhance efficacy of EGFR/PI3K inhibitors.Our results demonstrate that LSD1 protein expression can be modulated by stimulation of EGFR in patient-derived GSCs. We observed an increase in LSD1 protein expression following the addition of EGF in GSC 17 cells. The concurrent inhibition of EGFR/PI3K and LSD1 enhanced the in vitro efficacy compared to single agent, supporting convergence of kinase signaling and LSD1 dependent pathways. In fact, several treatment combinations of EGFR/PI3K inhibitors and LSD1 inhibitors resulted in synergistic effects in multiple GSC lines.In summary, our results highlight the need for effective therapy combinations that can reduce the population of GSCs and avoid the adaptive resistance that is typical of kinase inhibitors. Future studies will focus on evaluating the efficacy and tolerability of the most promising treatment combinations in vivo using orthotopic xenograft models of the GSCs. Citation Format: Lea Stitzlein, Matthew Luetzen, Caitlin McCabe, Maninder Khosla, Melissa Singh, Xiaoping Su, Yue Lu, Joy Gumin, Frederick Lang, Christopher Whitehead, Judith Sebolt-Leopold, Joya Chandra. Efficacy of EGFR/PI3K signaling inhibition is enhanced with LSD1 inhibition in glioblastoma stem cell (GSC) models [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 3329.

Abstract 5418: Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations

Abstract Introduction & Purpose: “HER2low breast cancer” is an operational term identifying breast carcinomas (BCs) displaying a HER2 score 1+ or score 2+ in immunohistochemistry (IHC) and lacking HER2 amplification. This paradigm shift in HER2 assessment in BC stems from substantial antitumor activity of potent antibody-drug conjugates (ADCs) targeting the HER2 receptor on the tumor cell membrane. Whether these tumors constitute a distinct pathological entity is a matter of debate. Experimental procedures: To dissect the biology of HER2low BCs we performed a high throughput mutational analysis coupled with gene expression profiling on 98 HER2low BCs [34 cases score 1+ (1), 15 cases score 2+ HER2 not amplified (2N), 49 cases score 2+ with HER2 copy numbers in the equivocal range (2E)], of which 89 were estrogen receptor (ER)-positive, and 9 ER-negative. DNA and RNA were extracted and subjected to targeted DNA sequencing (Illumina TSO500 panel) and targeted gene expression analysis (NanoString BC360 panel). Mutation rates were compared with in silico data from 1317 samples of the MSK Breast Cancer cohort (PMID: 30205045). Results: The most frequently mutated genes were PIK3CA (33/98, 33%), GATA3 (18/98, 18%), TP53 (17/98, 17%), HER2 (8/98, 8%, private to 2E cases). Tumor mutational burden mean values were significantly higher in score 1+ compared to score 2+ cases (p= 0.04), with a single score 1+ tumor harboring microsatellite instability (MSI). Mutational signatures 1, 5 and 13 (APOBEC) were the most prevalent, with signature 30 being private to HER2 score 1+ cases. Comparative statistics revealed mutational drift of HER2low BCs from both HER2 negative (19 differentially mutated genes) and HER2 positive BCs (11 differentially mutated genes). Potentially actionable genetic alterations (OncoKB classes) accounted for 43 SNVs (predominantly HER2 and PIK3CA mutations) and 17 CNVs (e.g. MET amplification and PTEN homozygous deletions), including a MSI tumor, distributed over 47 independent patients.Subgroup stratifications based on gene expression were observed when considering genes involved in angiogenesis, stromal marker and cytokines signalling. HER2 mRNA levels were significantly higher in 2E versus 2N and in 2E versus 1. Differential gene expression Kegg pathway analysis identified RTK activation restricted to 2E cases. Conclusions: HER2low BCs harbour a different mutational landscape from ER- and grade- matched HER2 positive and HER2 negative BCs. Differences across IHC score classes within HER2low carcinomas were demonstrated, with the 2E category showing distinct features (private HER2 mutations, HER2 mRNA levels significantly higher compared to the other groups, RTK pathway activation). Of note, besides the option of ADCs we demonstrate that up to 48% of cases may be targeted with different approaches exploiting actionable alterations. Citation Format: Enrico Berrino, Laura Annaratone, Ivana Sarotto, Maria Cecilia Zecchillo, Laura Casorzo, Riccardo Ponzone, Filippo Montemurro, Anna Sapino, Caterina Marchio. Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations [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 5418.

Intrinsically disordered regions couple the ligand binding and kinase activation of Trk neurotrophin receptors

SummaryReceptor tyrosine kinases (RTKs) are key players in development and several diseases. Understanding the molecular mechanism of RTK activation by its ligand could lead to the design of new RTK inhibitors. How the extracellular domain is coupled to the intracellular kinase domain is a matter of debate. Ligand-induced dimerization and ligand-induced conformational change of pre-formed dimers are two of the most proposed models. Recently we proposed that TrkA, the RTK for nerve growth factor (NGF), is activated by rotation of the transmembrane domain (TMD) pre-formed dimers upon NGF binding. However, one of the unsolved issues is how the ligand binding is conformationally coupled to the TMD rotation if unstructured extracellular juxtamembrane (eJTM) regions separate them. Here we use nuclear magnetic resonance in bicelles and functional studies to demonstrate that eJTM regions from the Trk family are intrinsically disordered and couple the ligand-binding domains and TMDs possibly via the interaction with NGF.

Abstract 2445: Alternative splicing dictates receptor tyrosine kinase fates in glioblastoma

Abstract Receptor tyrosine kinase (RTK) genes are frequently amplified and/or overexpressed in glioblastoma (GBM). RTK signaling can undergo two fates - attenuation via lysosomal degradation or prolonged signaling via recycling. Annexin A7 (ANXA7) is a membrane binding protein belonging to the Annexin family, presumed to participate in the endocytic pathway. ANXA7 is alternatively spliced (AS) by PTBP1, a ribonucleoprotein, and expressed as two isoforms - unspliced Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). We determined that I1 and I2 have divergent functional effects on RTK fates in GBM – I1 sorts RTKs for degradation thereby attenuating signaling, while I2 preferentially recycles RTKs and potentiates signaling. We hypothesize that I1, by virtue of its cassette exon, has the motifs necessary to target multiple RTKs for degradation thereby diminishing tumorigenic signaling in GBM.We generated multiple GBM cell lines that express endogenous I2 (I2 cells), or endogenous I2 + exogenous I1 (I1 cells). We evaluated the influence of I1 and/or I2 expression on RTK levels, activation and downstream events using western blot and co-immunoprecipitation assays. Receptor trafficking was observed using proximity ligation and immunofluorescence (IF) assays tagging different components of the endocytic pathway. To elucidate the motifs required for I1-mediated sorting, we used site-directed mutagenesis and created truncated I1 mutants lacking domains deemed necessary for degradation. We observed upregulated levels of several tumorigenic RTKs such as EGFR, MET, PDGFRα and EGFRvIII in the I2 cells. In I1 cells, however, we observed reduced levels and activation of all these RTKs along with diminished activation of downstream pathways. Immunostaining with endocytic markers revealed that RTKs were rapidly trafficked to the lysosomes in I1 cells indicative of degradation. Conversely, in I2 cells, RTKs sorted to the recycling endosomes perpetuating signaling. In our truncated I1 mutant models, we observed significantly higher levels of activated and total EGFR in a mutant with a 11 amino acid deletion in the cassette exon region. IF analysis showed that EGFR colocalized with recycling endosomes in this truncated mutant indicating that the absence of this domain impairs sorting to the lysosomes and re-routes the receptor to the recycling pathway causing sustained signaling. Dysregulated AS allows tumors to alter the transcriptome in favor of isoforms that drive tumor progression. Our results show that AS of ANXA7 can dictate the fate of RTK signaling by regulating the interaction of I1 and I2 with divergent endocytic processes in GBM. Domains encoded by the cassette exon, essential for I1-mediated RTK attenuation, are spliced out leading to unchecked signaling and tumor progression. Together, these results demonstrate how AS regulates protein interactions and signal transduction at a molecular level in GBM. Citation Format: Sindhu Nair, Rajani Rajbhandari, Centdrika Hurt, Markus Bredel. Alternative splicing dictates receptor tyrosine kinase fates in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2445.

The Role of Wild-Type RAS in Oncogenic RAS Transformation.

The RAS family of oncogenes (HRAS, NRAS, and KRAS) are among the most frequently mutated protein families in cancers. RAS-mutated tumors were originally thought to proliferate independently of upstream signaling inputs, but we now know that non-mutated wild-type (WT) RAS proteins play an important role in modulating downstream effector signaling and driving therapeutic resistance in RAS-mutated cancers. This modulation is complex as different WT RAS family members have opposing functions. The protein product of the WT RAS allele of the same isoform as mutated RAS is often tumor-suppressive and lost during tumor progression. In contrast, RTK-dependent activation of the WT RAS proteins from the two non-mutated WT RAS family members is tumor-promoting. Further, rebound activation of RTK–WT RAS signaling underlies therapeutic resistance to targeted therapeutics in RAS-mutated cancers. The contributions of WT RAS to proliferation and transformation in RAS-mutated cancer cells places renewed interest in upstream signaling molecules, including the phosphatase/adaptor SHP2 and the RasGEFs SOS1 and SOS2, as potential therapeutic targets in RAS-mutated cancers.

Topology-Switching Membrane Peptides for Determination of the Active Transmembrane Dimer Interface of the Epha2 Receptor

The EphA2 receptor tyrosine kinase is an interesting target for cancer therapeutics, as its rogue activation promotes tumor growth and metastasis. EphA2 activation is sensitive to dimerization induced by its single transmembrane (TM) domain. We thus previously designed the pH-sensitive TYPE7 peptide, which selectively activates EphA2 by binding its TM domain (Alves et al. eLife, 2018). TYPE7 comprises the TM sequence of EphA2, but with Glutamate residues added onto one helical face. To study the mechanism of TYPE7-induced EphA2 activation, we systematically rotated the Glu-containing helical interface. We performed biophysical and cell biology techniques on the six resulting peptides and molecular dynamics (MD) simulations on two, to study their effect on TM binding and activation of EphA2. First, we studied the interaction of the peptides with the membrane region of EphA2 reconstituted in lipid vesicles. We observed that the peptides are helical in the membrane, and most of them interact with EphA2. However, several peptides failed to activate EphA2 in cells, as assessed by EphA2-mediated cell migration. Next, we performed MD simulations to understand why similar peptides affect EphA2 activation differently. We observed that an activating peptide stabilizes the EphA2 TM dimer in a glycine zipper conformation. In contrast, a non-activating peptide stabilizes a complex in which the EphA2 helices are separated in a non-native, not-activation-competent conformation. The MD results suggest a mechanism for TYPE7-induced EphA2 activation, where peptide binding stabilizes the more active TM conformation. This provides support for the ligand-induced rotation hypothesis for RTK activation, where different helical interfaces correspond to active and inactive states. Additionally, these studies provide a basis for the specificity of TYPE7, since changing the available TM interface results in an EphA2 TM conformation that is less activation-competent.

Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics

In responses to activation of receptor tyrosine kinases (RTKs), crucial cell fate decisions depend on the duration and dynamics of ERK signaling. In PC12 cells, epidermal growth factor (EGF) induces transient ERK activation that leads to cell proliferation, whereas nerve growth factor (NGF) promotes sustained ERK activation and cell differentiation. These differences have typically been assumed to reflect distinct feedback mechanisms in the Raf-MEK-ERK signaling network, with the receptors themselves acting as simple upstream inputs. We failed to confirm the expected differences in feedback type when investigating transient versus sustained signaling downstream of the EGF receptor (EGFR) and NGF receptor (TrkA). Instead, we found that ERK signaling faithfully followed RTK dynamics when receptor signaling was modulated in different ways. EGFR activation kinetics, and consequently ERK signaling dynamics, were switched from transient to sustained when receptor internalization was inhibited with drugs or mutations, or when cells expressed a chimeric receptor likely to have impaired dimerization. In addition, EGFR and ERK signaling both became more sustained when substoichiometric levels of erlotinib were added to reduce duration of EGFR kinase activation. Our results argue that RTK activation kinetics play a crucial role in determining MAP kinase cascade signaling dynamics and cell fate decisions, and that signaling outcome can be modified by activating a given RTK in different ways.

Abstract 6304: Decrypting EGFR signaling with BRET biosensors: A novel approach to study RTK mutations and the effects of inhibitors

Abstract Receptor tyrosine kinases (RTKs) comprise nodes at the center of complex signaling networks regulating various aspects of cell growth, differentiation and survival. Dysregulated RTK activity can alter many cellular processes, often culminating in cancer. Consequently, RTKs are prime targets for new anti-cancer agents. Yet, the efficacy of these drugs is limited by the development of drug-induced adverse events and acquired resistance. Discovery of novel therapeutics devoid of such limitations requires a deeper understanding of often overlooked determinants of therapeutic efficacy, including kinetics, subcellular localization, signaling bias and drug target mutations. In this study, we present a live-cell BRET-based biosensor platform applicable to studying such key elements, which allows for real-time spatiotemporal monitoring of RTK signaling across more than 10 effector proteins/pathways. Using EGFR and two of its ligands (EGF and Epiregulin) as a model system, we used our platform to highlight spatiotemporal signaling bias. Indeed, Epiregulin-induced engagement of effector protein Grb2 was more efficacious (25%) and displayed faster kinetics relative to that observed with EGF. Interestingly, EGF, but not Epiregulin, promoted a gradual increase in Grb2 levels at early endosomes. These data highlight opportunities for the identification of biased ligands displaying improved therapeutic efficacy. The impact of various glioblastoma-related EGFR mutations on receptor signaling were also profiled. The EGFR(vI) mutant showed constitutive activity on the PLCG1 effector pathway while the EGFR(vV) mutant was uniquely constitutive on SHIP1 and SHIP2 pathways. Further, EGFR(vIII) was devoid of constitutive activity on some effector proteins (PLCG1, Grb2) and highly constitutive on others (PI3K, SHIP1/2). The G598V mutant showed constitutive activity on all pathways except Grb2. This data shows how oncogenic mutations can rewire an RTK's signaling network. Identification and targeting of common signaling nodes exploited by various mutants could enhance drug efficacy and minimize mutation-related drug resistance. Finally, RTK biosensors were used to screen for tyrosine kinase inhibitor (TKI) activity on EGFR kinase domain mutants found in NSCLC (T790M and C797S). 1st generation TKI Gefitinib and 4th generation TKI Osimertinib reversed EGF activation of WT EGFR. However, only Osimertinib could reverse EGF-mediated activation of EGFR(T790M) but was ineffective at inhibiting EGFR(C797S). The platform could thus serve as a tool to assess anti-cancer treatment efficacy against various RTK mutants. These data position our biosensor platform as a versatile technology to study multiple facets of RTK biology and pharmacology. Access to real-time, spatiotemporal readouts across multiple signaling pathways will enable the development of novel RTK-based anti-cancer agents. Citation Format: Florence Gross, Guilhem Dugast, Arturo D. Mancini, Stephan Schann, Xavier Leroy. Decrypting EGFR signaling with BRET biosensors: A novel approach to study RTK mutations and the effects of inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6304.