PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression

Summary: The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1 to program chromatin and ER transcriptional output. : Toska, Castel, et al. show that the PI3K pathway propagates its effects to control chromatin and estrogen receptor (ER) function through SGK1, a PI3K effector. PI3K inhibitors, via a negative-feedback loop, activate SGK1, which phosphorylates the histone lysine methyltransferase KMT2D to attenuate its activity and regulate ER response. Keywords: SGK1, KMT2D, PI3K pathway, estrogen receptor, breast cancer, chromatin regulation, AKT, PI3K inhibitor

TRK Fusions Are Enriched in Cancers with Uncommon Histologies and the Absence of Canonical Driver Mutations

TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion-positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion-positive cancers. Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. We identified 76 cases with confirmed TRK fusions (0.28% overall prevalence) involving 48 unique rearrangements and 17 cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers ( < 0.001) and lower tumor mutation burden ( < 0.001), with the exception of colorectal cancer where TRK fusions cooccur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months [95% confidence interval (CI), 4.8-13.2]. The best overall response rate achieved with chemotherapy containing-regimens across all lines of therapy was 63% (95% CI, 41-81). Among 12 patients treated with checkpoint inhibitors, a patient with MSI-H colorectal cancer had the only observed response. TRK fusion-positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors

Abstract 16: Landscape and outcome of TRK fusion-positive Cancers

Abstract TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion-positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, clinical outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion-positive cancers. Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with identified TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. In total, we identified 76 cases with confirmed TRK fusions (0.27% overall prevalence) involving 48 unique rearrangements and 17 distinct cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (p=4.4E-7) and lower tumor mutation burden (p=4.2E-9), with the exception of colorectal cancer where TRK fusions co-occur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months (95% CI: 4.8-13.2). The best ORR achieved with chemotherapy containing-regimens across all lines of therapy was 63% (95% CI: 41-81). Among 12 patients treated with checkpoint inhibitors, the only response observed was in an MSI-H colorectal patient. TRK fusion-positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors. Citation Format: Ezra Y. Rosen, Debra A. Goldman, Jaclyn F. Hechtman, Ryma Benayed, Alison M. Schram, Emiliano Cocco, Sophie Shifman, Yixiao Gong, Ritika Kundra, James P. Solomon, Alberto Bardelli, Maurizio Scaltriti, Alexander Drilon, Alexia Iasonos, Barry S. Taylor, David M. Hyman. Landscape and outcome of TRK fusion-positive Cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 16

Abstract 5680: TRK xDFG mutations trigger a sensitivity switch from type I to II kinase inhibitors

Abstract Background: TRK inhibition is the standard of care for patients with TRK fusion-positive solid tumors. TRK kinase domain mutations that impair drug binding are common mechanisms of resistance to 1st-generation TRK inhibitors. While 2nd-generation TRK inhibitors were designed to maintain kinase inhibition in this setting, the resistance to these agents is still poorly characterized. Methods and Results: We sequenced paired tumor biopsies and serial cell-free DNA (cfDNA) collected before therapy and at progression from patients treated with 2nd-generation TRK inhibitors (selitrectinib or repotrectinib). We identified 5 cases in which the acquisition of xDFG (G667) TRKA mutations was associated with resistance. Two patients whose tumors carried these substitutions pre-selitrectinib never responded to therapy, while three additional cases acquired these mutations upon progression to either selitrectinib or repotrectinib. In-silico molecular modeling combined with molecular dynamic simulations predicted that TRKA xDFG substitutions can confer resistance to 2nd-generation TRK inhibitors by generating steric hindrance that compromises drug binding. Accordingly, in vitro kinase assays showed that the IC50 for selitrectinib of TRKA xDFG mutants was >12 to >8000 fold higher compared to the IC50 of either TRKA wild type or the selitrectinib-sensitive TRKA G595R solvent front mutant. Interestingly, our data also suggest that TRKA xDFG substitutions induce conformational changes that stabilize the inactive (xDFG-out) conformation of the kinase, thus sensitizing it to type II inhibition. In vitro microscale thermophoresis revealed that the binding affinity of type II TRK inhibitors (cabozantinib or foretinib) to the TRKA G667C-mutant kinase was 8-10-fold higher compared to the type I inhibitor selitrectinib. We then tested the efficacy of type II TRK inhibitors against TRKA xDFG mutants in different cell models. A Bcan-Ntrk1-driven mouse model knocked in by CRISPR Cas9 to express the xDFG mutations was sensitive to type II but not to type I TRK inhibitors. Similar results were obtained using an LMNA-NTRK1-positive colorectal cell line that acquired the G667C substitution upon chronic selitrectinib treatment. Type II TRK inhibitor therapy achieved complete and durable responses also in patient-derived models with TRKA xDFG-mediated resistance to type I 2nd-generation agents. Conclusions: Our study uncovers a molecular switch induced by xDFG mutations that limits the sensitivity to type I kinase inhibitors by conformational changes that favor the inactive xDFG-out kinase state. This same switch in turn sensitizes these mutant kinases to type II inhibitors that effectively engage this inactive conformation. These results provide a paradigm for the rational development of 3rd-generation TKIs that address the problem of conformational resistance in tumors that are driven by oncogenic kinases. Citation Format: Emiliano Cocco, Ji Eun Lee, Srinivasaraghavan Kannan, Alison M. Schram, Helen H. Won, Sophie Shifman, Amanda Kulick, Laura Baldino, Eneda Toska, Sabrina Arena, Benedetta Mussolin, Ram Kannan, Neil Vasan, Alexander N. Gorelick, Michael F. Berger, Yi Liao, Uwe Rix, Alberto Bardelli, Jacklyn Hechtman, Elisa de Stanchina, David M. Hyman, Chandra Verma, Andrea Ventura, Alexander Drilon, Maurizio Scaltriti. TRK xDFG mutations trigger a sensitivity switch from type I to II kinase 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 5680

Abstract LB-118: Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation

Abstract Background: TRK inhibition is now standard of care for advanced pediatric and adult patients (pts) with TRK fusion solid tumors, regardless of origin. To date, TRK kinase domain mutations are the only known resistance mechanism, and next-generation TRK inhibitors active against these mutations such as LOXO-195 are being developed. We reasoned some pts will develop TRK-independent resistance and hypothesized that these pts will require unique therapeutic approaches. Methods: Paired tumor biopsies and serial cell-free DNA (cfDNA) prospectively collected from pts with TRK fusion-positive cancers treated with first- and next-generation TRK inhibitors before treatment and at progression were sequenced. In parallel, pt-derived and engineered models were analyzed. Results: Alterations involving upstream non-TRK receptor kinases and downstream MAPK pathway members were initially identified in tumors from 3 TRK fusion-positive gastrointestinal (GI) cancer pts who developed resistance to TRK inhibitors. Pt 1 with CTRC-NTRK1 pancreatic cancer developed temporally distinct emergent BRAF V600E and KRAS G12D mutations. Pt 2 with LMNA-NTRK1 colorectal cancer developed temporally distinct KRAS G12A and G12D mutations. Pt 3 with PLEKHA6-NTRK1 cholangiocarcinoma developed focal MET amplification. Phenocopying these clinical observations, pt-derived xenografts and primary cell lines developed BRAF and KRAS mutations following chronic TRK inhibition. Consistently, ectopic expression of these alterations conferred resistance to TRK inhibitors. Given that all 3 index pts had GI cancers, we expanded serial cfDNA sequencing to 5 additional TRK fusion-positive GI disease, identifying 3 with emergent MAPK alterations at progression, bringing the overall frequency of acquired MAPK alterations in GI cancers analyzed to 75% (6/8). To further evaluate whether these emergent alterations induced functional dependence on ERK signaling, pts 1-3 were treated with agents targeting these emergent alterations (dabrafenib + trametinib, LOXO-195 + trametinib, and LOXO-195 + crizotinib, respectively). Pt 1 achieved transient tumor regression, followed by outgrowth of KRAS-mutant disease. Pt 3 achieved a 4.5 months tumor regression. Sequencing at progression in pt 3 identified multiple acquired MET point mutations known to interfere with crizotinib binding. Conclusions: These data suggest that a subset of TRK fusion-positive cancers will develop off-target mechanisms of resistance to TRK inhibition. Relative to other TRK fusion-positive tumors, GI cancers may have a higher propensity for developing these bypass alterations that demonstrate remarkable convergence on ERK signaling. A portion of these mechanisms may be managed with simultaneous targeting of the TRK and MAPK pathways, although additional modeling is required to determine if upfront treatment would confer more durable responses. Citation Format: Emiliano Cocco, Amanda Kulick, Sandra Misale, Rona Yaeger, Pedram Razavi, Helen H. Won, Ryan Ptashkin, Jaclyn F. Hechtman, Eneda Toska, James Cownie, Romel Somwar, Sophie Shifman, Marissa Mattar, S Duygu Selçuklu, Aliaksandra Samoila, Sean Guzman, Brian B. Tuch, Kevin Ebata, Elisa de Stanchina, Rebecca J. Nagy, Richard B. Lanman, Michael F. Berger, Marc Ladanyi, David M. Hyman, Alexander Drilon, Maurizio Scaltriti, Alison M. Schram. Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-118

Neratinib is effective in breast tumors bearing both amplification and mutation of ERBB2 (HER2)

Mutations in , the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not amplified) tumors but are rare in "HER2-positive" ( amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of /HER2

The evolution of RET inhibitor resistance in RET-driven lung and thyroid cancers

The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors

On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. SIGNIFICANCE: In TRK fusion-positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation