Search

PDF file - 75K, This file contains detailed descriptions of materials and methods that had to be omitted from the main manuscript due to space limitations

Complete Data for Genetic Analysis of ctDNA and Tumor Tissue for All Samples.

PDF file - 59K, This file contains additional experimental data relating to the profile of JAA120, secretome screening results, gene expression data, and in vivo MET/FGR inhibitor synergy

Supplementary Figure S1. Confirmation of secondary point mutation in FGFR2-ZMYM4 allele. Supplementary Figure S2. Homology between FGFR2 and FGFR3 proteins. Supplementary Figure S3. FGFR2-OPTN fusion detected in all autopsy lesions. Supplementary Figure S4. FoundationOne analysis of autopsy lesions. Supplementary Figure S5. PTEN loss of heterozygosity (LOH) identified in Patient #2. Supplementary Figure S6. Mutational analysis of original resection specimen from Patient #2.

PDF file - 310K, This file contains additional experimental data relating to ligand-mediated rescue and drug combinations in cancer cell lines

XLS file - 147K, Expression of selected RTKs and cognate ligands in cancer cell lines as a basis for selecting models for testing of FGFR/MET inhibitor combinations

Purpose:The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection.Experimental Design:The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models.Results:Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations.Conclusions:Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

SUPPLEMENTARY APPENDIX TABLES

Purpose:Resistance to treatment with endocrine therapy in patients with HR+, HER2− advanced breast cancer (ABC) is common and dual inhibition of CDK4/6 and PI3K pathways may delay the development of resistance. This phase Ib trial evaluates the safety and tolerability of triple and double regimens containing the CDK4/6 inhibitor ribociclib.Patients and Methods:In this open-label, multicenter, phase Ib study, 70 postmenopausal women with HR+, HER2− ABC were enrolled into one of four treatment combinations: ribociclib (once daily, 3 weeks on, 1 week off) plus fulvestrant; ribociclib (continuous dosing) plus fulvestrant; ribociclib plus alpelisib plus fulvestrant; or ribociclib plus buparlisib plus fulvestrant.Results:The recommended phase II dose (RP2D) of ribociclib was confirmed to be 600 mg (3 weeks on, 1 week off) and 400 mg (continuous dosing) plus fulvestrant 500 mg. For the triple combination with buparlisib, the RP2D was ribociclib 400 mg plus buparlisib 30 mg plus fulvestrant 500 mg. Enrollment for the triple combinations was stopped due to unexpected toxicity. No RP2D was determined for the alpelisib combination. The safety profiles of the ribociclib plus fulvestrant combinations were consistent with those in previous studies. There was no marked difference in ribociclib exposure in the presence of triple-combination partners. The highest overall response rate was seen in the buparlisib triple combination (25.0%; 95% confidence interval, 9.8–46.7).Conclusions:Ribociclib plus fulvestrant demonstrated safety in the treatment of patients with HR+, HER2− ABC. Triple combinations with alpelisib or buparlisib plus fulvestrant are not recommended for phase II investigation.See related commentary by Clark et al., p. 371

Supplementary text and figures Supplementary Figure 1 A, visualization of the "inflection point" (or EC50) and "Amax" parameters that are shown in Fig. 2A. B, HGF mRNA expression (by RNA-seq) vs HGF protein in culture supernatant for 76 cancer cell lines. Cell lines with high HGF mRNA expression generally led to increased protein levels in supernatant. U87-MG and IM95 are labeled, because in vivo efficacy data are presented for those models. U87-MG shows a remarkably high level of HGF protein secretion despite relatively low HGF mRNA. C, labeled cell lines scored as hits with at least 2 out of 4 MET inhibitors, each screened twice across a subset of the CCLE. Hits were defined as Amax {less than or equal to} â^'25% for all compounds, and inflection point {less than or equal to} 100 nmol/L for capmatinib, {less than or equal to} 1 ï�­mol/L for crizotinib and JNJ-38877605, and {less than or equal to} 500 nmol/L for PF-4217903. The upper panel shows MET vs HGF mRNA expression, while the lower panel shows MET expression vs MET copy number for the same cell lines. Three regions that among themselves contain all hits (amplified, overexpression, autocrine) were defined such that the hit with the lowest value for the respective expression or copy number value sets the cut-off. Number of hits compared to total number of cell lines in the respective regions are indicated in brackets. Colors as in Figure 2. D, Time course of MET phosphorylation after a 5 minute pulse treatment with 100 ng/mL recombinant HGF in 2 cell lines. NCI-H596 cells bear a MET exon14 skipping mutation while A549 cells do not. Supplementary Figure 2 A, MET dependency (x axis) vs MET gene copy number (y axis), downloaded from https://depmap.org. MET gene dependency was estimated by applying the DEMETER2 model to a combination of 3 large-scale RNAi screens (Broad Achilles, Novartis DRIVE, Marcotte et al.) (7). B, MET dependency (x axis) vs HGF mRNA expression by RNA-seq (y axis), downloaded from https://depmap.org. MET gene dependency was determined from pooled CRISPR screening data (Avana 1.0 library, Broad Institute) applying the CERES algorithm (8,9). Supplementary Figure 3 A, potency comparison of several clinical MET inhibitors in cellular proliferation assays with EBC-1 cells. Representative dose-response curves are shown on the left, numerical inflection point values (mean {plus minus} standard deviation, n = 3) are displayed on the right. B, MET mRNA expression measured by Affymetrix human genome U133 Plus 2.0 arrays (x axis) or RNA-seq (y axis) in 67 lung cancer PDX models (Supplementary Table 3). Identifiers of the 3 models with highest expression are indicated. C, same models as in B, but displaying MET mRNA by RNA-seq (x axis) vs MET copy number by Affymetrix SNP 6.0 array (y axis). D, data as in Fig. 3B but showing individual tumor volumes under treatment with capmatinib. E, mouse body weights corresponding to the experiment shown in Fig. 3B. F, data as in Fig. 3C but phospho- MET and total MET values from multi-spot ELISA shown separately. G, repeat of capmatinib efficacy study with lung PDX tumors bearing a MET exon 14 skipping mutation (model LU5381) as in Fig. 3D, but longer treatment duration. An average regression of ~60% was observed on day 12. A vehicle control was not repeated in this study. H, antitumor efficacy of capmatinib (dosed as indicated) against xenografts of the gastric cancer cell line IM95, which expresses HGF. Supplementary Figure 4 A, Loewe excess for data shown in Fig. 4C (upper panel), and % inhibition as well as Loewe excess for a combination matrix of capmatinib and the selective BCL2 inhibitor venetoclax (lower panel), in the cell line NCI-H1993. Percent dead cells were quantified by dual imaging with propidium iodide (PI) and Hoechst 33342. B, Percent dead cells by PI/Hoechst (upper panel) and Loewe excess (lower panel) for EBC-1 cells treated as in Fig. 4C and Supplementary Fig. 4A. C, Loewe excess for data shown in Fig. 4D, cell lines as indicated. Here, data are based on a CellTiter-Glo readout with quantification of seeded cells at time of compound addition ("growth inhibition" calculation). Loewe excess also refers to the "growth inhibition" data. D, treatment of EBC-1 cells with the indicated dose matrix of capmatinib and erlotinib. Experimental setup and analysis as in Fig. 4D (docetaxel combination). Supplementary Figure 5 A, HCC827 or HCC827 GR lung cancer cells were exposed to gefitinib, capmatinib, or combinations in a fixed ratio for 72 hours before measuring cell viability using a resazurin assay. The x axis label corresponds to gefitinib concentrations, while capmatinib was used at 10-fold lower concentrations due to its higher potency. In combination, gefitinib and capmatinib were mixed at a ratio of 10:1. B, HCC827 or NCI-H3255 cells were treated with a dilution series of gefitinib in the presence or absence of 50 ng/mL recombinant HGF. Cell viability was measured after 96 hours (HCC827) or 72 hours (NCIH3255) using a resazurin assay. The initial amount of viable cells was quantified at the time of compound addition (dashed line), and cell growth on the y axis is expressed as a multiple of this value. C, lysates of the lung cancer PDX model X-1787, either treated with vehicle or crizotinib, were incubated on a phospho-RTK array. Phospho-MET is clearly detectable in vehicle-treated lysate, suggesting that the high MET mRNA expression in this model leads to MET protein expression and activation. D, anti-tumor efficacy of crizotinib against X-1787 PDX tumors, characterized by presence of EML4-ALK and high MET expression. N = 4 per arm. E, RKO cells (BRAF V600E-mutant colorectal cancer cells secreting HGF) were exposed to capmatinib and fixed ratios of dabrafenib and trametinib in a dose matrix as indicated. After 3 days, viable cells were quantified by CellTiter-Glo. Inhibition is quantified in 2 different ways, without or with regard of the initial amount of viable cells at the time of compound addition (left and middle panel, respectively). Calculations are explained in detail in Materials and Methods. The right panel shows the Loewe excess for "% inhibition."

Supplementary Table 1. IC50 values for capmatinib obtained in BaF3 TRP-MET cells and mutant variants as indicated after 3-day proliferation assays with a resazurin readout. MET kinase domain mutations were either generated by site-directed mutagenesis or obtain by random mutagenesis and selection with another selective MET inhibitor (5). Supplementary Table 2. Numerical data that are graphically represented in Figure 2. Supplementary Table 3. Combined data from CCLE screens with 4 MET inhibitors (capmatinib, crizotinib, JNJ- 38877605, and PF-4217903). Related to Supplementary Fig. 1C. Supplementary Table 4. MET genomics data and additional model information for a collection of lung cancer PDX models downloaded from https://compendium.criver.com/. The Excel table is filtered down to those 66 models with complete data for MET mRNA (both Affymetrix and RNA-seq), MET mutations, and MET gene copy number, but other lung cancer models with partial data are also contained within the table. The 3 models that were used for capmatinib in vivo efficacy studies are highlighted in yellow.

Supplementary Figures 1-2, Tables 1-4, Methods from A Drug Resistance Screen Using a Selective MET Inhibitor Reveals a Spectrum of Mutations That Partially Overlap with Activating Mutations Found in Cancer Patients

Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-18.

MYC transcription factors are well-established drivers of human cancers but despite being amongst the most frequently altered oncogenes, no approved therapy targeting MYC-driven tumors has been developed to date. MYC-driven cancers are known to be addicted to protein translation. This addiction creates a dependency on critical components of the translational machinery providing in turn a unique opportunity for therapeutic intervention. We hypothesized that targeting the translation termination factor GSPT1, a key regulator of protein synthesis, would constitute a vulnerability for MYC-driven tumors. Herein we further describe MRT-2359 a potent, selective and orally bioavailable degrader of GSPT1. MRT-2359 was rationally designed using our QuEENTM discovery engine and optimized to achieve a profound and preferential antiproliferative activity in MYC-driven cell lines, such as high N- and L-MYC mRNA expressing non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) lines. In line with expectations, MRT-2359 activity is dependent on both CRBN and the GSPT1 G-loop degron. We further demonstrate using an inducible system that the sole expression of either N- or L-MYC is sufficient to sensitize initially resistant NSCLC cells to MRT-2359. These studies therefore establish a causal link between N- and L-MYC expression and sensitivity to MRT-2359. Unlike MRT-2359, agents targeting the protein translation initiation machinery or repressing MYC transcription (CDK9 inhibitor) failed to show such differential activity. Mechanistically, RiboSeq and polysome profiling revealed that treatment with MRT-2359 in the N- or L-MYC high cell lines induces ribosome stalling at the stop codon, increased monosomes and decreased polysomes. These changes are indicative of translational repression and were confirmed using puromycilation assays. Proteomics and RNAseq studies finally demonstrated a significant reduction in the total levels of N- or L-MYC leading in turn to the downmodulation of MYC target genes. Despite robust degradation of GSPT1, no marked effect was observed in these assays in low N- or L-MYC lines, confirming the selective activity of MRT-2359 in MYC-driven lung cancers. Last, the anti-tumor activity of MRT-2359 was assessed in >80 lung patient-derived xenografts (PDXs). MRT-2359 demonstrated preferential activity in N- and L-MYC high NSCLC and SCLC PDXs, including numerous instances of tumor regressions, when dosed orally daily or intermittently. Similar levels of anti-tumor activity were also observed in neuroendocrine lung cancer and lymphoma PDXs. Together these results warrant further investigations in the clinic. Oral MRT-2359 is currently in a Phase 1/2 clinical trial in selected cancer patients with MYC-driven NSCLC, SCLC, high grade neuroendocrine cancers and diffuse large B-cell lymphoma (NCT05546268). Citation Format: Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Maciej Cabanski, Lisa Cantagallo, Agustin Chicas, Qian Chen, Anna Diesslin, Christopher King, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Dave Peck, Carolina Perdomo Ortiz, Martin Schillo, Ambika Singh, Ralph Tiedt, Simone Tortoioli, Silvia Buonamici, Filip Janku, Owen Wallace, Bernhard Fasching. Development of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for the treatment of lung cancers with MYC-induced translational addiction [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 3449.

Purpose: Resistance to treatment with endocrine therapy in patients with HR+, HER2− advanced breast cancer (ABC) is common and dual inhibition of CDK4/6 and PI3K pathways may delay the development of resistance. This phase Ib trial evaluates the safety and tolerability of triple and double regimens containing the CDK4/6 inhibitor ribociclib. Patients and Methods: In this open-label, multicenter, phase Ib study, 70 postmenopausal women with HR+, HER2− ABC were enrolled into one of four treatment combinations: ribociclib (once daily, 3 weeks on, 1 week off) plus fulvestrant; ribociclib (continuous dosing) plus fulvestrant; ribociclib plus alpelisib plus fulvestrant; or ribociclib plus buparlisib plus fulvestrant. Results: The recommended phase II dose (RP2D) of ribociclib was confirmed to be 600 mg (3 weeks on, 1 week off) and 400 mg (continuous dosing) plus fulvestrant 500 mg. For the triple combination with buparlisib, the RP2D was ribociclib 400 mg plus buparlisib 30 mg plus fulvestrant 500 mg. Enrollment for the triple combinations was stopped due to unexpected toxicity. No RP2D was determined for the alpelisib combination. The safety profiles of the ribociclib plus fulvestrant combinations were consistent with those in previous studies. There was no marked difference in ribociclib exposure in the presence of triple-combination partners. The highest overall response rate was seen in the buparlisib triple combination (25.0%; 95% confidence interval, 9.8–46.7). Conclusions: Ribociclib plus fulvestrant demonstrated safety in the treatment of patients with HR+, HER2− ABC. Triple combinations with alpelisib or buparlisib plus fulvestrant are not recommended for phase II investigation. See related commentary by Clark et al., p. 371

The histone 3 lysine 79 (H3K79) methyltransferase (HMT) DOT1L is known to play a critical role for growth and survival of MLL-rearranged leukemia. Serendipitous observations during high-throughput drug screens indicated that the use of DOT1L inhibitors might be expandable to multiple myeloma (MM). Through pharmacologic and genetic experiments, we could validate that DOT1L is essential for growth and viability of a subset of MM cell lines, in line with a recent report from another team. In vivo activity against established MM xenografts was observed with a novel DOT1L inhibitor. In order to understand the molecular mechanism of the dependency in MM, we examined gene expression changes upon DOT1L inhibition in sensitive and insensitive cell lines and discovered that genes belonging to the endoplasmic reticulum (ER) stress pathway and protein synthesis machinery were specifically suppressed in sensitive cells. Whole-genome CRISPR screens in the presence or absence of a DOT1L inhibitor revealed that concomitant targeting of the H3K4me3 methyltransferase SETD1B increases the effect of DOT1L inhibition. Our results provide a strong basis for further investigating DOT1L and SETD1B as targets in MM.

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectopic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A structure-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing testing of the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds displaying good exposure in mouse and nanomolar inhibition of target gene expression in cells were obtained and tested in vivo.

Purpose: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. Experimental Design: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. Results: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. Conclusions: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

Publisher's Note: There is a Blood Commentary on this article in this issue.

Background Overcoming resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) in patients with KRAS wildtype (WT) metastatic colorectal cancer (mCRC) could help meet the needs of patients with limited treatment options. Methods In this phase 1b study, patients with N/KRAS WT, MET-positive mCRC who had progressed following anti-EGFR mAb treatment received escalating oral doses of capmatinib (150, 300, and 400 mg) twice daily plus weekly intravenous cetuximab (at the approved dose). The primary objective was to establish a recommended dose for expansion (RDE) of capmatinib in combination with cetuximab. Safety, preliminary activity, pharmacokinetics, and pharmacodynamics were also explored. Results Thirteen patients were enrolled. No patients experienced a dose-limiting toxicity at investigated doses; the RDE was established as capmatinib 400 mg twice daily plus cetuximab. All patients experienced adverse events (AEs) suspected to be related to the study treatment. Five patients (38.5%) reported study-drug–related AEs of grade 3/4 in severity. No patients achieved a complete or partial response according to RECIST v1.1; however, tumor shrinkage of 29–44% was observed in 4 patients. Conclusions Capmatinib plus cetuximab was well tolerated. Preliminary signs of activity were observed. Further investigation is warranted to obtain efficacy data and refine predictive biomarkers of response. Clinical trial registration NCT02205398.

This study presents strategies to render triple-negative breast cancers sensitive to CDK4/6 inhibitors., Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor–positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

Purpose To estimate the maximum tolerated dose (MTD) and/or identify the recommended Phase II dose (RP2D) for combined INC280 and buparlisib in patients with recurrent glioblastoma with homozygous phosphatase and tensin homolog (PTEN) deletion, mutation or protein loss. Methods This multicenter, open-label, Phase Ib/II study included adult patients with glioblastoma with mesenchymal-epithelial transcription factor (c-Met) amplification. In Phase Ib, patients received INC280 as capsules or tablets in combination with buparlisib. In Phase II, patients received INC280 only. Response was assessed centrally using Response Assessment in Neuro-Oncology response criteria for high-grade gliomas. All adverse events (AEs) were recorded and graded. Results 33 patients entered Phase Ib, 32 with altered PTEN. RP2D was not declared due to potential drug–drug interactions, which may have resulted in lack of efficacy; thus, Phase II, including 10 patients, was continued with INC280 monotherapy only. Best response was stable disease in 30% of patients. In the selected patient population, enrollment was halted due to limited activity with INC280 monotherapy. In Phase Ib, the most common treatment-related AEs were fatigue (36.4%), nausea (30.3%) and increased alanine aminotransferase (30.3%). MTD was identified at INC280 Tab 300 mg twice daily + buparlisib 80 mg once daily. In Phase II, the most common AEs were headache (40.0%), constipation (30.0%), fatigue (30.0%) and increased lipase (30.0%). Conclusion The combination of INC280/buparlisib resulted in no clear activity in patients with recurrent PTEN-deficient glioblastoma. More stringent molecular selection strategies might produce better outcomes. Trial registration: NCT01870726.

Inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) is associated with robust antitumor activity. Ribociclib (LEE011) is an orally bioavailable CDK4/6 inhibitor that is approved for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer, in combination with an aromatase inhibitor, and is currently being evaluated in several additional trials. Here, we report the preclinical profile of ribociclib. When tested across a large panel of kinase active site binding assays, ribociclib and palbociclib were highly selective for CDK4, while abemaciclib showed affinity to several other kinases. Both ribociclib and abemaciclib showed slightly higher potency in CDK4-dependent cells than in CDK6-dependent cells, while palbociclib did not show such a difference. Profiling CDK4/6 inhibitors in large-scale cancer cell line screens in vitro confirmed that RB1 loss of function is a negative predictor of sensitivity. We also found that routinely used cellular viability assays measuring adenosine triphosphate levels as a proxy for cell numbers underestimated the effects of CDK4/6 inhibition, which contrasts with assays that assess cell number more directly. Robust antitumor efficacy and combination benefit was detected when ribociclib was added to encorafenib, nazartinib, or endocrine therapies in patient-derived xenografts.

Background: The objectives of this phase II study were to determine the clinical activity of the MET tyrosine kinase inhibitor capmatinib (INC280) in patients with MET-dysregulated advanced hepatocellular carcinoma (HCC) and to assess the safety, pharmacokinetics, and correlation of biomarkers with the response. Methods: This phase II, open-label, single-arm study evaluated twice daily (BID) oral capmatinib in a dose-determining stage, utilizing a Bayesian Logistic Regression Model (BLRM) subject to Escalation with Overdose Control criteria, safety, pharmacokinetics, and pharmacodynamic information to determine a recommended dose for expansion (RDE) evaluating efficacy in patients with MET-dysregulated HCC. Results: A total of 38 patients received treatment. In the dose-determining stage, patients received capmatinib 300 mg BID capsules ( n = 8), and in the expansion, patients received 600 mg BID capsules ( n = 28) or 400 mg BID tablets ( n = 2) based on the BLRM and other relevant clinical data. No predefined qualifying adverse events (AEs) were observed during the first 28 days of treatment, and the RDE was 600 mg BID capsules (equivalent pharmacokinetics to 400 mg BID tablets). The most common any causality AEs were nausea (42%), vomiting (37%), and diarrhea (34%). In the expansion stage, in a subgroup of 10 patients with MET-high HCC, the overall response rate was 30%, including 1 durable complete response (>600 days) and 2 partial responses [1 durable (>600 days)]. Conclusions: Single agent capmatinib at the RDE is tolerable with a manageable safety profile. Antitumor activity was seen in a subset of patients with MET-dysregulated (MET-high) HCC. Trial registration: ClinicalTrials.gov: NCT01737827. https://clinicaltrials.gov/ct2/show/NCT01737827

Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemias. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identification of 7, a highly potent, selective and structurally novel Dot1L inhibitor.

[Image: see text] In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectopic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A structure-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing testing of the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds displaying good exposure in mouse and nanomolar inhibition of target gene expression in cells were obtained and tested in vivo.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2.

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.Leukemia advance online publication, 3 January 2017; doi:10.1038/leu.2016.327.

Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermethylation of H3K79 and misexpression of genes (including HoxA), which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was cocrystallized with DOT1L and optimized using structure-based ligand optimization to yield compound 8 (IC50 = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a pocket adjacent to the SAM binding site.

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.

Rearrangements in the mixed lineage leukemia (MLL) gene define a distinct, aggressive form of acute leukemia with poor prognosis. The MLL gene encodes for a SET domain histone methyl transferase that catalyzes the methylation of histone 3 lysine 4 (H3K4) at specific gene loci, thus regulating transcription of developmental genes including HOX genes. In disease-linked translocations, the catalytic SET domain is lost and the remaining part fused to a variety of partners, e.g. AF4, AF9, AF10 and ENL. These MLL fusion proteins directly interact with disruptor of telomeric silencing 1-like protein (DOT1L), the only known H3K79 methyltransferase. The H3K79me2 mark is broadly associated with active transcription. Mislocated enzymatic activity of DOT1L causes local H3K79 hypermethylation, misexpression of leukomogenic genes, e.g. HOXA9 and MEIS1, and the aberrant maintenance of a stem cell-like state. Current treatment options of MLL are limited to chemotherapy and allogeneic hematopoietic stem cell transplantation, and outcomes remain poor. The first and only clinical DOT1L inhibitor Pinometostat (EPZ-5676), an S-Adenosyl Methionine (SAM) competitive inhibitor, showed only modest activity and emerging resistance in adult acute leukemia. Pinometostat is no oral drug, but requires administration as continuous infusion to achieve sufficient exposure and sustained target inhibition. So far, there is no approved DOT1L inhibitor and the need remains to develop effective DOT1L inhibitors. We report the identification of new SAM-competitive, structurally SAM-unrelated DOT1L inhibitors with subnanomolar biochemical potency. Compounds with nanomolar cellular activity and good exposure in mouse were tested in tumor xenograft models. Compounds 8 and 9 showed excellent blood exposure after a single dose. However, repeated dosing led to reduced exposure due to cytochrome P450 (Cyp450) 3A4 induction, insufficient pharmacodynamics (PD) and lack of efficacy. Further modification resulted in compound 10 that could be administered orally and was stable upon repeated dosing. A 300 mg/kg dose covered efficacious blood exposure for 24 h, but was not tolerated by tumor-bearing mice. Unfortunately, a 6-fold reduced dose did not achieve sufficient PD modulation and efficacy. Additional activities led to compound 11, which lost its oral bioavailability and had to be administered subcutaneously. Nevertheless, compound 11 achieved tumor growth inhibition in the MV4-11 and Molm-13 xenograft models in mice. In conclusion, we progressed in making DOT1L inhibitors with improved PK properties, but further optimization is required to generate a viable clinical candidate. The limited efficacy obtained with compound 11 and lack of efficacy observed with subcutaneous administration of EPZ-5676 around the maximally tolerated dose illustrate the difficulty to achieve a sustained level of DOT1L inhibitor in vivo to suppress DOT1L activity sufficiently. Furthermore, H3K79me2 and efficacy in vivo seem disconnected. Citation Format: Andreas Weiss, Frederic Stauffer, Clemens Clemens, Henrik Möbitz, Christian Ragot, Kim S. Beyer, Keith Calkins, Claudio Thoma, Daniel Guthy, Michael Kiffe, Bernard Van Eerdenbrugh, William R. Sellers, Francesco Hofmann, Ralph Tiedt, Christoph Gaul. A new DOT1L inhibitor with in vivo activity in mouse models of MLL-translocated leukemia [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 1770.

BACKGROUND c-MET and PI3K pathways are dysregulated in GBM and can drive cancer growth in pre-clinical models. INC280 is a small molecule inhibitor of c-MET; BKM120 is a pan-class I PI3K inhibitor. A phase Ib/II study was designed to evaluate INC280+BKM120 in patients with recurrent GBM ({"type":"clinical-trial","attrs":{"text":"NCT01870726","term_id":"NCT01870726"}}NCT01870726).

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion–positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the FGFR2 kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intralesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation led to BGJ398 resistance and was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary FGFR2 mutations represent an important clinical resistance mechanism that may guide the development of future therapeutic strategies. Significance: We report the first genetic mechanisms of clinical acquired resistance to FGFR inhibition in patients with FGFR2 fusion–positive ICC. Our findings can inform future strategies for detecting resistance mechanisms and inducing more durable remissions in ICC and in the wide variety of cancers where the FGFR pathway is being explored as a therapeutic target. Cancer Discov; 7(3); 252–63. ©2016 AACR. See related commentary by Smyth et al., p. 248. This article is highlighted in the In This Issue feature, p. 235

The overall power of kinase inhibitors is substantially overshadowed by the acquisition of drug resistance. To address this issue, we systematically assessed the potential of secreted proteins to induce resistance to kinase inhibitors. To this end, we developed a high-throughput platform for screening a cDNA library encoding 3,432 secreted proteins in cellular assays. Using cancer cells originally dependent on either MET, FGFR2, or FGFR3, we observed a bypass of dependence through ligand-mediated activation of alternative receptor tyrosine kinases (RTK). Our findings indicate a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other. We further provide evidence that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect. Significance: Although initial tumor responses to kinase inhibitors can be significant, therapeutic benefit is often limited by the emergence of resistance (e.g., as a consequence of mutations in the drug target or through activation of alternative pathways to bypass dependence on the original target). Because the activation of alternative growth-promoting kinases by stimulation with their cognate ligands can constitute such a bypass mechanism, the identification of growth factors as possible mediators of resistance to kinase inhibitors is of clinical interest. Cancer Discov; 2(10); 948–59. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 857.

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes. Cancer Res; 71(15); 5255–64. ©2011 AACR.

We generated mice expressing a full-length Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl−/− background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wild-type controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpl-transgenic Mpl−/− mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count.

Background Hereditary thrombocythemia is an autosomal dominant disorder with clinical features resembling sporadic essential thrombocythemia. Germline mutations in families with hereditary thrombocythemia have been identified in the gene for thrombopoietin ( TPHO ) and its receptor, MPL. Design and Methods Here we characterized a THPO mutation in a hereditary thrombocythemia pedigree with 11 affected family members. Results Affected family members carry a G → C transversion in the splice donor of intron 3 of THPO that co-segregated with thrombocytosis within the pedigree. We previously described the identical mutation in a Dutch family with hereditary thrombocythemia. Haplotype analysis using single nucleotide polymorphisms surrounding the mutation indicated that the mutations arose independently in the two families. MPL protein levels, but not mRNA levels, were low in platelets from affected family members. Bone marrow histology showed features compatible with those of essential thrombocythemia, but the megakaryocytes were unusually compact, as assessed by planimetric analysis. Impaired microcirculation resulting in brief episodes of fainting and dizziness that responded well to aspirin were the predominant clinical features in a total of 23 affected family members studied. Disease onset is earlier in patients with hereditary thrombocythemia than in those with essential thrombocythemia, but the frequencies of thrombotic, vascular and hemorrhagic events are similar in the two groups. Conclusions A mutation in THPO occurred de novo in the same position as in a previously described family with hereditary thrombocythemia. Patients with this mutation have elevated serum levels of thrombopoietin and a phenotype that responds to aspirin and does not require cytoreductive treatment.

An acquired somatic mutation in the JAK2 gene (JAK2-V617F) is present in the majority of patients with myeloproliferative disorders (MPDs). Several phenotypic manifestations (polycythemia vera [PV], essential thrombocythemia [ET], and primary myelofibrosis) can be associated with the same mutation. We generated JAK2-V617F transgenic mice using a human JAK2 gene with the sequences encoding the kinase domain placed in the inverse orientation and flanked by antiparallel loxP sites. Crossing mice of one transgenic line (FF1) with transgenic mice expressing Cre-recombinase under the control of the hematopoiesis specific Vav promoter led to expression of JAK2-V617F that was lower than the endogenous wild-type Jak2. These mice developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia. Induction of the JAK2-V617F transgene with the interferon-inducible MxCre resulted in expression of JAK2-V617F approximately equal to wild-type Jak2 and a PV-like phenotype with increased hemoglobin, thrombocytosis, and neutrophilia. Higher levels of JAK2-V617F in mouse bone marrow by retroviral transduction caused a PV-like phenotype without thrombocytosis. These data are consistent with the hypothesis that the ratio of mutant to wild-type JAK2 is critical for the phenotypic manifestation. A similar correlation was also found in patients with MPD.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9Δ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin β3 phosphorylation and PtdIns(3,4)P2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9Δ platelets were still able to extend lamellipodia but without stress fiber–like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl3-induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outside-in signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9Δ mice.

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin alphaIIbbeta3-mediated platelet aggregation and beta1 integrin-mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet beta1 and beta3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian alpha2beta1 and alphaIIbbeta3 integrins in vivo.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in a broad range of cancers and occur in ~20% of ICCs. As seen with other targeted therapies, however, acquired resistance has limited the efficacy of selective FGFR kinase inhibitors such as BGJ398. In a phase II trial of patients with advanced refractory cholangiocarcinoma harboring an FGFR gene alteration, BGJ398 displayed an overall response rate of 22%, but the durability of response was short in some patients. We report the molecular basis of acquired resistance in 4 patients with advanced FGFR2-fusion positive ICC via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), the primary tumor, and metastases. Each patient enjoyed an initial response, but all subsequently progressed within 10 months. Serial analysis of cfDNA revealed multiple point mutations in the FGFR2 kinase domain at progression (Table 1). The gatekeeper mutation, p. V564F, sterically hinders drug binding and was identified in 3 of 4 patients. In patient #1, five different FGFR2 mutations were detected in the post-progression cfDNA but only one, p. K641R, was identified in the post-progression biopsy. A rapid autopsy was performed, and genomic characterization of 12 metastatic lesions revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance that was surmountable by structurally distinct FGFR inhibitors. Thus, our report provides the first genetic evidence of clinical acquired resistance to FGFR inhibitor therapy in patients and informs future strategies for detecting mechanisms of resistance and promoting more durable remissions. Clinical and Molecular Data on Patients with Advanced Refractory CCA treated with BGJ398Patient IDSexAge at Diagnosis (years)FGFR2 Fusion partnerMaximum ResponseProgression Free Survival on BGJ398 (months)Overall Survival since Diagnosis (months)FGFR2 Mutations on cfDNA at progression1F47OPTN28%3.632V564F, N549H, K641R, E565A, L617V2F59ZMYMY450%5.631V564F, N549H/K, E565A, K659M3F69SORBS168%12.650K659M, K714R4M43BICC137%7.418V564F Note: This abstract was not presented at the meeting. Citation Format: Lipika Goyal, Supriya K. Saha, Leah Y. Liu, Giulia Siravegna, Ignaty Leshchiner, Leanne G. Ahronian, Jochen K. Lennerz, Phuong Vu, Benedetta Mussolin, Stephanie Reyes, Pascal Furet, A. John Iafrate, Gad Getz, Diana G. Porta, Ralph Tiedt, Alberto Bardelli, Dejan Juric, Ryan B. Corcoran, Nabeel Bardeesy, Andrew X. Zhu. Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in FGFR2 fusion-positive cholangiocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2017-4114