David J. Konieczkowski

A Melanoma Cell State Distinction Influences Sensitivity to MAPK Pathway Inhibitors

UNLABELLED Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma. SIGNIFICANCE Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.

Mutations in zebrafish leucine-rich repeat-containing six-like affect cilia motility and result in pronephric cysts, but have variable effects on left-right patterning.

Cilia defects have been implicated in a variety of human diseases and genetic disorders, but how cilia motility contributes to these phenotypes is still unknown. To further our understanding of how cilia function in development, we have cloned and characterized two alleles of seahorse, a zebrafish mutation that results in pronephric cysts. seahorse encodes Lrrc6l, a leucine-rich repeat-containing protein that is highly conserved in organisms that have motile cilia. seahorse is expressed in zebrafish tissues known to contain motile cilia. Although mutants do not affect cilia structure and retain the ability to interact with Disheveled, both alleles of seahorse strongly affect cilia motility in the zebrafish pronephros and neural tube. Intriguingly, although seahorse mutations variably affect fluid flow in Kupffers vesicle, they can have very weak effects on left-right patterning. Combined with recently published results, our alleles suggest that the function of seahorse in cilia motility is separable from its function in other cilia-related phenotypes.

Characterizing genomic alterations in cancer by complementary functional associations

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.

Fine-mapping in African Americans of 8 recently discovered genetic loci for plasma lipids: The Jackson Heart Study

Background—Genome-wide association studies in cohorts of European descent have identified novel genomic regions as associated with lipids, but their relevance in African Americans remains unclear. Methods and Results—We genotyped 8 index single nucleotide polymorphisms (SNPs) and 488 tagging SNPs across 8 novel lipid loci in the Jackson Heart Study, a community-based cohort of 4605 African Americans. For each trait, we calculated residuals adjusted for age, sex, and global ancestry and performed multivariable linear regression to detect genotype-phenotype association with adjustment for local ancestry. To explore admixture effects, we conducted stratified analyses in individuals with a high probability of 2 African ancestral alleles or at least 1 European allele at each locus. We confirmed 2 index SNPs as associated with lipid traits in African Americans, with suggestive association for 3 more. However, the effect sizes for 4 of the 5 associated SNPs were larger in the European local ancestry subgroup compared with the African local ancestry subgroup, suggesting that the replication is driven by European ancestry segments. Through fine-mapping, we discovered 3 new SNPs with significant associations, 2 with consistent effect on triglyceride levels across ancestral groups: rs636523 near DOCK7/ANGPTL3 and rs780093 in GCKR. African linkage disequilibrium patterns did not assist in narrowing association signals. Conclusions—We confirm that 5 genetic regions associated with lipid traits in European-derived populations are relevant in African Americans. To further evaluate these loci, fine-mapping in larger African American cohorts and/or resequencing will be required.

Resistance to EGFR blockade in colorectal cancer: liquid biopsies and latent subclones.

Two recent papers identify KRAS activation as a mechanism of acquired resistance to EGFR blockade in colorectal cancer. In doing so, they suggest that resistance to single-agent EGFR blockade will be unavoidable because these alterations exist as latent subclones within the tumor even prior to the initiation of therapy.

A Convergence-Based Framework for Cancer Drug Resistance

Despite advances in cancer biology and therapeutics, drug resistance remains problematic. Resistance is often multifactorial, heterogeneous, and prone to undersampling. Nonetheless, many individual mechanisms of targeted therapy resistance may coalesce into a smaller number of convergences, including pathway reactivation (downstream re-engagement of original effectors), pathway bypass (recruitment of a parallel pathway converging on the same downstream output), and pathway indifference (development of a cellular state independent of the initial therapeutic target). Similar convergences may also underpin immunotherapy resistance. Such parsimonious, convergence-based frameworks may help explain resistance across tumor types and therapeutic categories and may also suggest strategies to overcome it.

Abstract PR04: A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors

The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAF V600E -mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAF V600 -mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAF V600 -mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naive patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAF V600 -mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAF V600 -mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.

Abstract C221: A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene.

Oncogenic mutations in BRAF are found in 50-70% of malignant melanomas and in ∼8% of all cancers. Activating BRAF mutations confer exquisite dependency on RAF/MEK/ERK signaling and RAF and MEK inhibitors achieved striking clinical response in the majority of patients with BRAFV600E-mutant melanoma. However, nearly all patients progress to therapeutic resistance and there are limited treatment options for patients with relapsed tumors. Identification of genes that are essential only in the presence of BRAFV600E mutation may inform alternative as well as combinatorial therapeutic approaches. Here, we used systematic RNA interference to discover synthetic lethal interactions in melanoma cells carrying BRAFV600E mutation. In addition, Project Achilles at the Broad Institute reported systematic loss-of-function studies in more than 100 human cancer cell lines. We intersected the list of genes that are synthetic lethal with BRAFV600E mutation in our melanoma study and that of Project Achilles. As a result, we have identified novel “druggable” targets for cells driven by BRAFV600E mutation. Our extensive in vitro and in vivo validation has confirmed and established key synthetic lethal interactions with BRAFV600E mutation. One major mechanism of acquired resistance to RAF and MEK inhibition is reactivation of MEK/ERK pathway. Importantly, we demonstrated that these synthetic lethal genes are also essential in the context of acquired resistance to RAF and MEK inhibition. Together, our study has identified novel targets for the treatment of oncogenic BRAF-driven melanoma and suggests targeting these genes as novel therapeutic strategies to overcome acquired resistance to RAF and MEK inhibition. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C221. Citation Format: Chengyin Min, David J. Konieczkowski, Christine Kwon, Marika Linja, Krishna Vasudevan, Barbara Weir, Eva Goetz, Levi Garraway. A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C221.

Fine-Mapping in African Americans of 8 Recently Discovered Genetic Loci for Plasma LipidsClinical Perspective: The Jackson Heart Study

Background—Genome-wide association studies in cohorts of European descent have identified novel genomic regions as associated with lipids, but their relevance in African Americans remains unclear. Methods and Results—We genotyped 8 index single nucleotide polymorphisms (SNPs) and 488 tagging SNPs across 8 novel lipid loci in the Jackson Heart Study, a community-based cohort of 4605 African Americans. For each trait, we calculated residuals adjusted for age, sex, and global ancestry and performed multivariable linear regression to detect genotype-phenotype association with adjustment for local ancestry. To explore admixture effects, we conducted stratified analyses in individuals with a high probability of 2 African ancestral alleles or at least 1 European allele at each locus. We confirmed 2 index SNPs as associated with lipid traits in African Americans, with suggestive association for 3 more. However, the effect sizes for 4 of the 5 associated SNPs were larger in the European local ancestry subgroup compared with the African local ancestry subgroup, suggesting that the replication is driven by European ancestry segments. Through fine-mapping, we discovered 3 new SNPs with significant associations, 2 with consistent effect on triglyceride levels across ancestral groups: rs636523 near DOCK7/ANGPTL3 and rs780093 in GCKR. African linkage disequilibrium patterns did not assist in narrowing association signals. Conclusions—We confirm that 5 genetic regions associated with lipid traits in European-derived populations are relevant in African Americans. To further evaluate these loci, fine-mapping in larger African American cohorts and/or resequencing will be required.

Fine-Mapping in African Americans of 8 Recently Discovered Genetic Loci for Plasma LipidsClinical Perspective

Background—Genome-wide association studies in cohorts of European descent have identified novel genomic regions as associated with lipids, but their relevance in African Americans remains unclear. Methods and Results—We genotyped 8 index single nucleotide polymorphisms (SNPs) and 488 tagging SNPs across 8 novel lipid loci in the Jackson Heart Study, a community-based cohort of 4605 African Americans. For each trait, we calculated residuals adjusted for age, sex, and global ancestry and performed multivariable linear regression to detect genotype-phenotype association with adjustment for local ancestry. To explore admixture effects, we conducted stratified analyses in individuals with a high probability of 2 African ancestral alleles or at least 1 European allele at each locus. We confirmed 2 index SNPs as associated with lipid traits in African Americans, with suggestive association for 3 more. However, the effect sizes for 4 of the 5 associated SNPs were larger in the European local ancestry subgroup compared with the African local ancestry subgroup, suggesting that the replication is driven by European ancestry segments. Through fine-mapping, we discovered 3 new SNPs with significant associations, 2 with consistent effect on triglyceride levels across ancestral groups: rs636523 near DOCK7/ANGPTL3 and rs780093 in GCKR. African linkage disequilibrium patterns did not assist in narrowing association signals. Conclusions—We confirm that 5 genetic regions associated with lipid traits in European-derived populations are relevant in African Americans. To further evaluate these loci, fine-mapping in larger African American cohorts and/or resequencing will be required.