Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here, we observe that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via genetic evolution of initially T790M-negative drug tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells had a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR; treatment with navitoclax, an inhibitor of BCL-XL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Highlights d Cancer-associated fibroblasts contribute to pancreatic cancer heterogeneity d Cancer cells can have a double-positive phenotype: proliferation and invasion d High CAF abundance linked with DP cells enriched for MAPK and STAT3 co-signaling d Intra-tumoral gland types provide tissue heterogeneity linked with clinical outcome

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the signifi cant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the fi rst selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.

SummaryPersonalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.

Evolved resistance to tyrosine kinase inhibitor (TKI) targeted therapies remains a major clinical challenge. In EGFR mutant non-small cell lung cancer (NSCLC), failure of EGFR TKIs can result from both genetic and epigenetic mechanisms of acquired drug resistance. Widespread reports of histologic and gene expression changes consistent with an epithelial-to-mesenchymal transition (EMT) have been associated with initially surviving drug tolerant persister cells, which can seed bona fide genetic mechanisms of resistance to EGFR TKIs. While therapeutic approaches targeting fully resistant cells, such as those harboring an EGFR T790M mutation, have been developed, a clinical strategy for preventing the emergence of persister cells remains elusive. Using mesenchymal cell lines derived from biopsies of patients who progressed on EGFR TKI as surrogates for persister populations, we performed whole-genome CRISPR screening and identified FGFR1 as the top target promoting survival of mesenchymal EGFR mutant cancers. Although numerous previous reports of FGFR signaling contributing to EGFR TKI resistance in vitro exist, the data has not yet been sufficiently compelling to instigate a clinical trial testing this hypothesis, nor has the role of FGFR in promoting the survival of persister cells been elucidated. In this study, we find that combining EGFR and FGFR inhibitors inhibited the survival and expansion of EGFR mutant drug tolerant cells over long time periods, preventing the development Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The combination of CDK4/6 inhibitors with antiestrogen therapies signifi cantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN , through increased AKT activation, was suffi cient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these fi ndings provide critical insight into how this single genetic event may cause clinical crossresistance to multiple targeted therapies in the same patient, with implications for optimal treatmentsequencing strategies. SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as fi rst-line treatment for ER-positive advanced breast cancer. Importantly, these fi ndings have near-term clinical relevance because PTEN loss also limits the effi cacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multi-kinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant ‘gatekeeper’ mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.