Publications by authors named "Deepak Sampath"

91 Publications

Machine-Learning and Chemicogenomics Approach Defines and Predicts Cross-Talk of Hippo and MAPK Pathways.

Cancer Discov 2021 Mar 18;11(3):778-793. Epub 2020 Nov 18.

Department of Discovery Oncology, Genentech, Inc., South San Francisco, California.

Hippo pathway dysregulation occurs in multiple cancers through genetic and nongenetic alterations, resulting in translocation of YAP to the nucleus and activation of the TEAD family of transcription factors. Unlike other oncogenic pathways such as RAS, defining tumors that are Hippo pathway-dependent is far more complex due to the lack of hotspot genetic alterations. Here, we developed a machine-learning framework to identify a robust, cancer type-agnostic gene expression signature to quantitate Hippo pathway activity and cross-talk as well as predict YAP/TEAD dependency across cancers. Further, through chemical genetic interaction screens and multiomics analyses, we discover a direct interaction between MAPK signaling and TEAD stability such that knockdown of YAP combined with MEK inhibition results in robust inhibition of tumor cell growth in Hippo dysregulated tumors. This multifaceted approach underscores how computational models combined with experimental studies can inform precision medicine approaches including predictive diagnostics and combination strategies. SIGNIFICANCE: An integrated chemicogenomics strategy was developed to identify a lineage-independent signature for the Hippo pathway in cancers. Evaluating transcriptional profiles using a machine-learning method led to identification of a relationship between YAP/TAZ dependency and MAPK pathway activity. The results help to nominate potential combination therapies with Hippo pathway inhibition..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0706DOI Listing
March 2021

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-X Inhibitor.

ACS Med Chem Lett 2020 Oct 30;11(10):1829-1836. Epub 2020 Mar 30.

Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-X inhibitor that selectively and potently induces apoptosis in BCL-X-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-X inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-X. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549103PMC
October 2020

Venetoclax Increases Intratumoral Effector T Cells and Antitumor Efficacy in Combination with Immune Checkpoint Blockade.

Cancer Discov 2020 Sep 4. Epub 2020 Sep 4.

Translational Onco-logy, AbbVie Inc., North Chicago, Illinois.

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli and did not antagonize T-cell activation induced by anti-PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-X provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. SIGNIFICANCE: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency-approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.
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http://dx.doi.org/10.1158/2159-8290.CD-19-0759DOI Listing
September 2020

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

ACS Med Chem Lett 2020 Jun 26;11(6):1342-1347. Epub 2020 May 26.

Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of that showed ionic interaction of azetidine with Asp351 residue. Importantly, showed favorable metabolic stability and good oral exposure. exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294714PMC
June 2020

Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models.

Haematologica 2021 Apr 1;106(4):1034-1046. Epub 2021 Apr 1.

Department of Translational Oncology, Genentech, Inc., South San Francisco, CA, USA.

FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~25% of adult acute myeloid leukemia cases and are associated with poor prognosis. Venetoclax, a selective BCL-2 inhibitor, has limited monotherapy activity in relapsed/refractory acute myeloid leukemia with no responses observed in a small subset of FLT3-ITD+ patients. Further, FLT3-ITD mutations emerged at relapse following venetoclax monotherapy and combination therapy suggesting a potential mechanism of resistance. Therefore, we investigated the convergence of FLT3-ITD signaling on the BCL-2 family proteins and determined combination activity of venetoclax and FLT3-ITD inhibition in preclinical models. In vivo, venetoclax combined with quizartinib, a potent FLT3 inhibitor, showed greater anti-tumor efficacy and prolonged survival compared to monotherapies. In a patient-derived FLT3-ITD+ xenograft model, cotreatment with venetoclax and quizartinib at clinically relevant doses had greater anti-tumor activity in the tumor microenvironment compared to quizartinib or venetoclax alone. Use of selective BCL-2 family inhibitors further identified a role for BCL-2, BCL-XL and MCL-1 in mediating survival in FLT3-ITD+ cells in vivo and highlighted the need to target all three proteins for greatest anti-tumor activity. Assessment of these combinations in vitro revealed synergistic combination activity for quizartinib and venetoclax but not for quizartinib combined with BCL-XL or MCL-1 inhibition. FLT3-ITD inhibition was shown to indirectly target both BCL-XL and MCL-1 through modulation of protein expression, thereby priming cells toward BCL-2 dependence for survival. These data demonstrate that FLT3-ITD inhibition combined with venetoclax has impressive anti-tumor activity in FLT3-ITD+ acute myeloid leukemia preclinical models and provides strong mechanistic rational for clinical studies.
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http://dx.doi.org/10.3324/haematol.2019.244020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017817PMC
April 2021

Loss of glucocorticoid receptor expression mediates in vivo dexamethasone resistance in T-cell acute lymphoblastic leukemia.

Leukemia 2020 08 17;34(8):2025-2037. Epub 2020 Feb 17.

Department of Pediatrics, University of California, San Francisco, CA, USA.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.
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http://dx.doi.org/10.1038/s41375-020-0748-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440098PMC
August 2020

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Bioorg Med Chem Lett 2020 02 18;30(4):126907. Epub 2019 Dec 18.

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.
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http://dx.doi.org/10.1016/j.bmcl.2019.126907DOI Listing
February 2020

VCAM-1 Density and Tumor Perfusion Predict T-cell Infiltration and Treatment Response in Preclinical Models.

Neoplasia 2019 10 11;21(10):1036-1050. Epub 2019 Sep 11.

Biomedical Imaging, Genentech Inc., South San Francisco, CA. Electronic address:

Cancer immunotherapies have demonstrated durable responses in a range of different cancers. However, only a subset of patients responds to these therapies. We set out to test if non-invasive imaging of tumor perfusion and vascular inflammation may be able to explain differences in T-cell infiltration in pre-clinical tumor models, relevant for treatment outcomes. Tumor perfusion and vascular cell adhesion molecule (VCAM-1) density were quantified using magnetic resonance imaging (MRI) and correlated with infiltration of adoptively transferred and endogenous T-cells. MRI biomarkers were evaluated for their ability to detect tumor rejection 3 days after T-cell transfer. Baseline levels of these markers were used to assess their ability to predict PD-L1 treatment response. We found correlations between MRI-derived VCAM-1 density and infiltration of endogenous or adoptively transferred T-cells in some preclinical tumor models. Blocking T-cell binding to endothelial cell adhesion molecules (VCAM-1/ICAM) prevented T-cell mediated tumor rejection. Tumor rejection could be detected 3 days after adoptive T-cell transfer prior to tumor volume changes by monitoring the extracellular extravascular volume fraction. Imaging tumor perfusion and VCAM-1 density before treatment initiation was able to predict the response of MC38 tumors to PD-L1 blockade. These results indicate that MRI based assessment of tumor perfusion and VCAM-1 density can inform about the permissibility of the tumor vasculature for T-cell infiltration which may explain some of the observed variance in treatment response for cancer immunotherapies.
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http://dx.doi.org/10.1016/j.neo.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744528PMC
October 2019

Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Cell 2019 08 25;178(4):949-963.e18. Epub 2019 Jul 25.

Department of Translational Oncology, Genentech, South San Francisco, CA 94080, USA. Electronic address:

Estrogen receptor-positive (ER) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
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http://dx.doi.org/10.1016/j.cell.2019.06.026DOI Listing
August 2019

Convergent genetic aberrations in murine and human T lineage acute lymphoblastic leukemias.

PLoS Genet 2019 06 14;15(6):e1008168. Epub 2019 Jun 14.

Department of Pediatrics, University of California San Francisco, San Francisco, CA, United States of America.

The lack of predictive preclinical models is a fundamental barrier to translating knowledge about the molecular pathogenesis of cancer into improved therapies. Insertional mutagenesis (IM) in mice is a robust strategy for generating malignancies that recapitulate the extensive inter- and intra-tumoral genetic heterogeneity found in advanced human cancers. While the central role of "driver" viral insertions in IM models that aberrantly increase the expression of proto-oncogenes or disrupt tumor suppressors has been appreciated for many years, the contributions of cooperating somatic mutations and large chromosomal alterations to tumorigenesis are largely unknown. Integrated genomic studies of T lineage acute lymphoblastic leukemias (T-ALLs) generated by IM in wild-type (WT) and Kras mutant mice reveal frequent point mutations and other recurrent non-insertional genetic alterations that also occur in human T-ALL. These somatic mutations are sensitive and specific markers for defining clonal dynamics and identifying candidate resistance mechanisms in leukemias that relapse after an initial therapeutic response. Primary cancers initiated by IM and resistant clones that emerge during in vivo treatment close key gaps in existing preclinical models, and are robust platforms for investigating the efficacy of new therapies and for elucidating how drug exposure shapes tumor evolution and patterns of resistance.
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http://dx.doi.org/10.1371/journal.pgen.1008168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594654PMC
June 2019

Concomitant targeting of BCL2 with venetoclax and MAPK signaling with cobimetinib in acute myeloid leukemia models.

Haematologica 2020 03 23;105(3):697-707. Epub 2019 May 23.

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. , the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.
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http://dx.doi.org/10.3324/haematol.2018.205534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049339PMC
March 2020

Neutralization of BCL-2/X Enhances the Cytotoxicity of T-DM1 .

Mol Cancer Ther 2019 06 8;18(6):1115-1126. Epub 2019 Apr 8.

Department of Cell Biology and Ludwig Center at Harvard, Harvard Medical School, Boston, Massachusetts.

One of the most recent advances in the treatment of HER2 breast cancer is the development of the antibody-drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Because anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/X would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/X via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model, which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked amplification, suggesting that BCL-2/X inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/X blockade and support clinical investigation of this combination beyond -amplified and/or HER2-overexpressed tumors.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6758547PMC
June 2019

Venetoclax plus R- or G-CHOP in non-Hodgkin lymphoma: results from the CAVALLI phase 1b trial.

Blood 2019 05 8;133(18):1964-1976. Epub 2019 Mar 8.

Université de Lille, CHU Lille, Groupe de Recherche sur les Formes Injectables et les Technologies Associées, Lille, France.

Novel strategies, such as chemosensitization with targeted agents, that build on the success of standard immunochemotherapy show promise for the treatment of non-Hodgkin lymphoma (NHL). Here, we report a phase 1b study investigating dose escalation of the BCL2 inhibitor, venetoclax, in combination with rituximab or obinutuzumab and cyclophosphamide, doxorubicin, vincristine, and prednisone (R-/G-CHOP) chemotherapy in B-cell NHL. Objectives included safety assessment and determination of a recommended phase 2 dose (RP2D). Fifty-six patients were enrolled, most with follicular lymphoma (43%) or diffuse large B-cell lymphoma (DLBCL; 32%). Dose-limiting toxicities were reported in 3/14 patients at the first venetoclax dose (200 mg/d), after which dosing was changed from daily to 10 days per cycle and escalated to 800 mg. A further reduction to 5 days per cycle occurred at the 800-mg dose level in the G-CHOP arm. Cytopenias were predominant among grade 3/4 events and reported at a higher rate than expected, particularly in the G-CHOP arm; however, safety was manageable. Overall response rates were 87.5% (R-CHOP and G-CHOP combinations); complete response (CR) rates were 79.2% and 78.1%, respectively. Most double-expressor (BCL2 and MYC) DLBCL patients (87.5%; n = 7/8) achieved CR. Although the maximum tolerated dose was not reached, the RP2D for venetoclax with R-CHOP was established at 800 mg days 4 to 10 of cycle 1 and days 1 to 10 of cycles 2 to 8; higher doses were not explored, and this dosing schedule demonstrated an acceptable safety profile. This regimen is subsequently being evaluated in first-line DLBCL in the phase 2 portion of the study. This trial was registered at www.clinicaltrials.gov as #NCT02055820.
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http://dx.doi.org/10.1182/blood-2018-11-880526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497517PMC
May 2019

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Bioorg Med Chem Lett 2019 04 1;29(7):905-911. Epub 2019 Feb 1.

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.
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http://dx.doi.org/10.1016/j.bmcl.2019.01.036DOI Listing
April 2019

Combined MEK and BCL-2/X Inhibition Is Effective in High-Grade Serous Ovarian Cancer Patient-Derived Xenograft Models and BIM Levels Are Predictive of Responsiveness.

Mol Cancer Ther 2019 03 24;18(3):642-655. Epub 2019 Jan 24.

Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School, Boston, Massachusetts.

Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/X inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in most models. , GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models-those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/X inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399746PMC
March 2019

The Kinase Activity of Hematopoietic Progenitor Kinase 1 Is Essential for the Regulation of T Cell Function.

Cell Rep 2018 10;25(1):80-94

Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target.
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http://dx.doi.org/10.1016/j.celrep.2018.09.012DOI Listing
October 2018

Preclinical assessment of the ADME, efficacy and drug-drug interaction potential of a novel NAMPT inhibitor.

Xenobiotica 2019 Sep 17;49(9):1063-1077. Epub 2019 Jan 17.

a Genentech, Inc., Drug Metabolism and Pharmacokinetics , South San Francisco , CA , USA.

GNE-617 (-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[1,2-]pyridine-6-carboxamide) is a potent, selective nicotinamide phosphoribosyltransferase (NAMPT) inhibitor being explored as a potential treatment for human cancers. Plasma clearance was low in monkeys and dogs (9.14 mL minkg and 4.62 mL minkg, respectively) and moderate in mice and rats (36.4 mL minkg and 19.3 mL minkg, respectively). Oral bioavailability in mice, rats, monkeys and dogs was 29.7, 33.9, 29.4 and 65.2%, respectively. Allometric scaling predicted a low clearance of 3.3 mL minkg and a volume of distribution of 1.3 L kg in human. Efficacy (57% tumor growth inhibition) in Colo-205 CRC tumor xenograft mice was observed at an oral dose of 15 mg/kg BID (AUC = 10.4 µM h). Plasma protein binding was moderately high. GNE-617 was stable to moderately stable . Main human metabolites identified in human hepatocytes were formed primarily by CYP3A4/5. Transporter studies suggested that GNE-617 is likely a substrate for MDR1 but not for BCRP. Simcyp simulations suggested a low (CYP2C9 and CYP2C8) or moderate (CYP3A4/5) potential for drug-drug interactions. The potential for autoinhibition was low. Overall, GNE-617 exhibited acceptable preclinical properties and projected human PK and dose estimates.
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http://dx.doi.org/10.1080/00498254.2018.1528407DOI Listing
September 2019

Pan-Cancer Metabolic Signature Predicts Co-Dependency on Glutaminase and De Novo Glutathione Synthesis Linked to a High-Mesenchymal Cell State.

Cell Metab 2018 09 28;28(3):383-399.e9. Epub 2018 Jun 28.

Translational Oncology, Genentech, South San Francisco, CA 94080, USA. Electronic address:

The enzyme glutaminase (GLS1) is currently in clinical trials for oncology, yet there are no clear diagnostic criteria to identify responders. The evaluation of 25 basal breast lines expressing GLS1, predominantly through its splice isoform GAC, demonstrated that only GLS1-dependent basal B lines required it for maintaining de novo glutathione synthesis in addition to mitochondrial bioenergetics. Drug sensitivity profiling of 407 tumor lines with GLS1 and gamma-glutamylcysteine synthetase (GCS) inhibitors revealed a high degree of co-dependency on both enzymes across indications, suggesting that redox balance is a key function of GLS1 in tumors. To leverage these findings, we derived a pan-cancer metabolic signature predictive of GLS1/GCS co-dependency and validated it in vivo using four lung patient-derived xenograft models, revealing the additional requirement for expression of GAC above a threshold (logRPKM + 1 ≥ 4.5, where RPKM is reads per kilobase per million mapped reads). Analysis of the pan-TCGA dataset with our signature identified multiple indications, including mesenchymal tumors, as putative responders to GLS1 inhibitors.
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http://dx.doi.org/10.1016/j.cmet.2018.06.003DOI Listing
September 2018

Cotargeting BCL-2 and PI3K Induces BAX-Dependent Mitochondrial Apoptosis in AML Cells.

Cancer Res 2018 06 20;78(11):3075-3086. Epub 2018 Mar 20.

Department of Internal Medicine, Virginia Commonwealth University and the Massey Cancer Center, Virginia.

Inhibitors targeting BCL-2 apoptotic proteins have significant potential for the treatment of acute myeloid leukemia (AML); however, complete responses are observed in only 20% of patients, suggesting that targeting BCL-2 alone is insufficient to yield durable responses. Here, we assessed the efficacy of coadministration of the PI3K/mTOR inhibitor GDC-0980 or the p110β-sparing PI3K inhibitor taselisib with the selective BCL-2 antagonist venetoclax in AML cells. Tetracycline-inducible downregulation of BCL-2 significantly sensitized MV4-11 and MOLM-13 AML cells to PI3K inhibition. Venetoclax/GDC-0980 coadministration induced rapid and pronounced BAX mitochondrial translocation, cytochrome c release, and apoptosis in various AML cell lines in association with AKT/mTOR inactivation and MCL-1 downregulation; ectopic expression of MCL-1 significantly protected cells from this regimen. Combined treatment was also effective against primary AML blasts from 17 patients, including those bearing various genetic abnormalities. Venetoclax/GDC-0980 markedly induced apoptosis in primitive CD34/38/123 AML cell populations but not in normal hematopoietic progenitor CD34 cells. The regimen was also active against AML cells displaying intrinsic or acquired venetoclax resistance or tumor microenvironment-associated resistance. Either combinatorial treatment markedly reduced AML growth and prolonged survival in a systemic AML xenograft mouse model and diminished AML growth in two patient-derived xenograft models. Venetoclax/GDC-0980 activity was partially diminished in BAK cells and failed to induce apoptosis in BAX and BAXBAK cells, whereas BIM cells were fully sensitive. Similar results were observed with venetoclax alone in and systemic xenograft models. Collectively, these studies demonstrate that venetoclax/GDC-0980 exhibits potent anti-AML activity primarily through BAX and, to a lesser extent, BAK. These findings argue that dual BCL-2 and PI3K inhibition warrants further evaluation in AML. Combined treatment with clinically relevant PI3K and BCL-2 inhibitors may prove effective in the treatment of acute myeloid leukemia. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984704PMC
June 2018

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

Chemistry 2018 Apr 25;24(19):4830-4834. Epub 2018 Mar 25.

WuXi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, P. R. China.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.
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http://dx.doi.org/10.1002/chem.201800859DOI Listing
April 2018

An Anti-GDNF Family Receptor Alpha 1 (GFRA1) Antibody-Drug Conjugate for the Treatment of Hormone Receptor-Positive Breast Cancer.

Mol Cancer Ther 2018 03 27;17(3):638-649. Epub 2017 Dec 27.

Genentech, Inc., 1 DNA Way, South San Francisco, California.

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both and The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0813DOI Listing
March 2018

Flavopiridol enhances ABT-199 sensitivity in unfavourable-risk multiple myeloma cells in vitro and in vivo.

Br J Cancer 2018 02 14;118(3):388-397. Epub 2017 Dec 14.

Division of Hematology/Oncology, Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.

Background: The BCL-2-specific BH3-mimetic ABT-199 (venetoclax) has been reported to be principally active against favourable-risk multiple myeloma (MM) cells, prompting efforts to extend its activity to include more resistant, higher-risk MM subsets.

Methods: Effects of the CDK9 inhibitor flavopiridol (FP; alvocidib) on responses to ABT-199 were examined in MM cells. Cell death and protein expression were evaluated by western blot and immunofluorescence. Xenograft models were used to study combination effects in vivo.

Results: FP synergistically increased ABT-199 lethality in both ABT-199-sensitive and insensitive MM cells. FP blocked CDK9 activation/positive transcription elongation factor B phosphorylation, downregulated MCL-1, increased BCL-2/MCL-1 ratios, and upregulated BIM. MCL-1 ectopic expression or knockdown in MM cells significantly diminished or increased ABT-199 sensitivity, respectively. CDK9 knockdown triggered MCL-1 downregulation and increased ABT-199 activity, whereas BIM knockdown significantly reduced FP/ABT-199 lethality. FP also enhanced ABT-199 lethality in unfavourable prognosis primary MM cells. HS-5 cell co-culture failed to protect MM cells from the FP/ABT-199 regimen, suggesting circumvention of microenvironmental signals. Finally, FP/ABT-199 significantly increased survival in systemic xenograft and immune-competent MM models while exhibiting minimal toxicity.

Conclusions: These findings argue that CDK9 inhibitors, for example, FP may increase the antimyeloma activity of ABT-199, including in unfavourable-risk MM minimally responsive to ABT-199 alone.
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http://dx.doi.org/10.1038/bjc.2017.432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808038PMC
February 2018

Found in Translation: How Preclinical Research Is Guiding the Clinical Development of the BCL2-Selective Inhibitor Venetoclax.

Cancer Discov 2017 12 16;7(12):1376-1393. Epub 2017 Nov 16.

Dana-Farber Cancer Institute, Boston, Massachusetts.

Since the discovery of apoptosis as a form of programmed cell death, targeting the apoptosis pathway to induce cancer cell death has been a high-priority goal for cancer therapy. After decades of effort, drug-discovery scientists have succeeded in generating small-molecule inhibitors of antiapoptotic BCL2 family proteins. Innovative medicinal chemistry and structure-based drug design, coupled with a strong fundamental understanding of BCL2 biology, were essential to the development of BH3 mimetics such as the BCL2-selective inhibitor venetoclax. We review a number of preclinical studies that have deepened our understanding of BCL2 biology and facilitated the clinical development of venetoclax. Basic research into the pathways governing programmed cell death have paved the way for the discovery of apoptosis-inducing agents such as venetoclax, a BCL2-selective inhibitor that was recently approved by the FDA and the European Medicines Agency. Preclinical studies aimed at identifying BCL2-dependent tumor types have translated well into the clinic thus far and will likely continue to inform the clinical development of venetoclax and other BCL2 family inhibitors.
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http://dx.doi.org/10.1158/2159-8290.CD-17-0797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728441PMC
December 2017

Monitoring and Targeting Anti-VEGF Induced Hypoxia within the Viable Tumor by F-MRI and Multispectral Analysis.

Neoplasia 2017 Nov 6;19(11):950-959. Epub 2017 Oct 6.

Department of Biomedical Imaging, Genentech Inc., South San Francisco, CA. Electronic address:

The effect of anti-angiogenic agents on tumor oxygenation has been in question for a number of years, where both increases and decreases in tumor pO have been observed. This dichotomy in results may be explained by the role of vessel normalization in the response of tumors to anti-angiogenic therapy, where anti-angiogenic therapies may initially improve both the structure and the function of tumor vessels, but more sustained or potent anti-angiogenic treatments will produce an anti-vascular response, producing a more hypoxic environment. The first goal of this study was to employ multispectral (MS) F-MRI to noninvasively quantify viable tumor pO and evaluate the ability of a high dose of an antibody to vascular endothelial growth factor (VEGF) to produce a strong and prolonged anti-vascular response that results in significant tumor hypoxia. The second goal of this study was to target the anti-VEGF induced hypoxic tumor micro-environment with an agent, tirapazamine (TPZ), which has been designed to target hypoxic regions of tumors. These goals have been successfully met, where an antibody that blocks both murine and human VEGF-A (B20.4.1.1) was found by MS F-MRI to produce a strong anti-vascular response and reduce viable tumor pO in an HM-7 xenograft model. TPZ was then employed to target the anti-VEGF-induced hypoxic region. The combination of anti-VEGF and TPZ strongly suppressed HM-7 tumor growth and was superior to control and both monotherapies. This study provides evidence that clinical trials combining anti-vascular agents with hypoxia-activated prodrugs should be considered to improved efficacy in cancer patients.
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http://dx.doi.org/10.1016/j.neo.2017.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635323PMC
November 2017

Systems analysis of apoptotic priming in ovarian cancer identifies vulnerabilities and predictors of drug response.

Nat Commun 2017 08 28;8(1):365. Epub 2017 Aug 28.

Department of Cell Biology, Ludwig Center at Harvard, Harvard Medical School, Boston, MA, 02115, USA.

The lack of effective chemotherapies for high-grade serous ovarian cancers (HGS-OvCa) has motivated a search for alternative treatment strategies. Here, we present an unbiased systems-approach to interrogate a panel of 14 well-annotated HGS-OvCa patient-derived xenografts for sensitivity to PI3K and PI3K/mTOR inhibitors and uncover cell death vulnerabilities. Proteomic analysis reveals that PI3K/mTOR inhibition in HGS-OvCa patient-derived xenografts induces both pro-apoptotic and anti-apoptotic signaling responses that limit cell killing, but also primes cells for inhibitors of anti-apoptotic proteins. In-depth quantitative analysis of BCL-2 family proteins and other apoptotic regulators, together with computational modeling and selective anti-apoptotic protein inhibitors, uncovers new mechanistic details about apoptotic regulators that are predictive of drug sensitivity (BIM, caspase-3, BCL-X) and resistance (MCL-1, XIAP). Our systems-approach presents a strategy for systematic analysis of the mechanisms that limit effective tumor cell killing and the identification of apoptotic vulnerabilities to overcome drug resistance in ovarian and other cancers.High-grade serous ovarian cancers (HGS-OvCa) frequently develop chemotherapy resistance. Here, the authors through a systematic analysis of proteomic and drug response data of 14 HGS-OvCa PDXs demonstrate that targeting apoptosis regulators can improve response of these tumors to inhibitors of the PI3K/mTOR pathway.
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http://dx.doi.org/10.1038/s41467-017-00263-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573720PMC
August 2017

Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer.

Clin Cancer Res 2017 Sep 12;23(18):5561-5572. Epub 2017 Jun 12.

Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, London, United Kingdom.

Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity. A panel of cell lines representative of TNBC was tested for and sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging. The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both ( < 0.001 LAR vs. basal-like) and in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in -mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes. Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175044PMC
September 2017

Phase I Dose-Escalation Study of Taselisib, an Oral PI3K Inhibitor, in Patients with Advanced Solid Tumors.

Cancer Discov 2017 07 22;7(7):704-715. Epub 2017 Mar 22.

Memorial Sloan Kettering Cancer Center, New York, New York.

Taselisib is a potent and selective tumor growth inhibitor through PI3K pathway suppression. Thirty-four patients with locally advanced or metastatic solid tumors were treated (phase I study, modified 3+3 dose escalation; 5 cohorts; 3-16 mg taselisib once-daily capsule). Taselisib pharmacokinetics were dose-proportional; mean half-life was 40 hours. Frequent dose-dependent, treatment-related adverse events included diarrhea, hyperglycemia, decreased appetite, nausea, rash, stomatitis, and vomiting. At 12 and 16 mg dose levels, dose-limiting toxicities (DLT) were observed, with an accumulation of higher-grade adverse events after the cycle 1 DLT assessment window. Pharmacodynamic findings showed pathway inhibition at ≥3 mg in patient tumor samples, consistent with preclinical -mutant tumor xenograft models. Confirmed response rate was 36% for -mutant tumor patients with measurable disease [5/14: 4 breast cancer (3 patients at 12 mg); 1 non-small cell lung cancer], where responses started at 3 mg, and 0% in patients with tumors without known hotspot mutations (0/15). Preliminary data consistent with preclinical data indicate increased antitumor activity of taselisib in patients with -mutant tumors (in comparison with patients with tumors without known activating hotspot mutations) starting at the lowest dose tested of 3 mg, thereby supporting higher potency for taselisib against -mutant tumors. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-1080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501742PMC
July 2017

KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer.

Cell 2017 02 16;168(5):817-829.e15. Epub 2017 Feb 16.

Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA; Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA. Electronic address:

Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in Kras "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
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http://dx.doi.org/10.1016/j.cell.2017.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541948PMC
February 2017