A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts.

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.

RAF-Mutant Melanomas Differentially Depend on ERK2 Over ERK1 to Support Aberrant MAPK Pathway Activation and Cell Proliferation.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. IMPLICATIONS: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

A Proof of Concept for Biomarker-Guided Targeted Therapy against Ovarian Cancer Based on Patient-Derived Tumor Xenografts.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Distinct Transcriptional Programming Drive Response to MAPK Inhibition in BRAF V600-Mutant Melanoma Patient-Derived Xenografts.

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAF V600-mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo, in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF-high, "epithelial-like" transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven "mesenchymal-like" transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF-high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAF V600-mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo, which in turn predicts their response to further pathway suppression using a combination of BRAF, MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAF V600-mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer.

Hyperactivation of MAPK Signaling Is Deleterious to RAS/RAF-mutant Melanoma.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

Tumor-derived IFN triggers chronic pathway agonism and sensitivity to ADAR loss.

Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases 1-4 . Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade 5-7 . Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.

The Lineage Determining Factor GRHL2 Collaborates with FOXA1 to Establish a Targetable Pathway in Endocrine Therapy-Resistant Breast Cancer.

Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins.

Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer.

Evolved resistance to tyrosine kinase inhibitor (TKI)-targeted therapies remains a major clinical challenge. In epidermal growth factor receptor (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 EGFRT790M 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 fibroblast growth factor receptor 1 (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 have 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 of fully resistant cancers in multiple vitro models and in vivo. These results suggest that dual EGFR and FGFR blockade may be a promising clinical strategy for both preventing and overcoming EMT-associated acquired drug resistance and provide motivation for the clinical study of combined EGFR and FGFR inhibition in EGFR-mutated NSCLCs.

BRAF-inhibitor Associated MEK Mutations Increase RAF-Dependent and -Independent Enzymatic Activity.

Alterations in MEK1/2 occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in BRAF-V600E-mutant melanoma and colorectal cancer. Efforts were undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant, MEK1-Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition.Implications: This study suggests that alternate modes of target inhibition, such as ERK inhibition, will be required to effectively treat tumors harboring these MEK1/2-resistant alleles. Mol Cancer Res; 15(10); 1431-44. ©2017 AACR.

Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models.

The efficacy of ALK inhibitors in patients with ALK-mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions.

UNLABELLED: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. SIGNIFICANCE: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.

A chemical genetics approach for the functional assessment of novel cancer genes.

Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.

Inhibition of Casein Kinase 1 Alpha Prevents Acquired Drug Resistance to Erlotinib in EGFR-Mutant Non-Small Cell Lung Cancer.

Patients with lung tumors harboring activating mutations in the EGF receptor (EGFR) show good initial treatment responses to the EGFR tyrosine kinase inhibitors (TKI) erlotinib or gefitinib. However, acquired resistance invariably develops. Applying a focused shRNA screening approach to identify genes whose knockdown can prevent and/or overcome acquired resistance to erlotinib in several EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, we identified casein kinase 1 α (CSNK1A1, CK1α). We found that CK1α suppression inhibits the NF-κB prosurvival signaling pathway. Furthermore, downregulation of NF-κB signaling by approaches independent of CK1α knockdown can also attenuate acquired erlotinib resistance, supporting a role for activated NF-κB signaling in conferring acquired drug resistance. Importantly, CK1α suppression prevented erlotinib resistance in an HCC827 xenograft model in vivo. Our findings suggest that patients with EGFR-mutant NSCLC might benefit from a combination of EGFR TKIs and CK1α inhibition to prevent acquired drug resistance and to prolong disease-free survival.

FGFR-Mediated Reactivation of MAPK Signaling Attenuates Antitumor Effects of Imatinib in Gastrointestinal Stromal Tumors.

UNLABELLED: Activating mutations in either KIT or PDGFRA are present in approximately 90% of gastrointestinal stromal tumors (GIST). Although treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients, the beneficial effect is not durable. Here, we report that ligands from the FGF family reduced the effectiveness of imatinib in GIST cells, and FGF2 and FGFR1 are highly expressed in all primary GIST samples examined. The combination of KIT and FGFR inhibition showed increased growth inhibition in imatinib-sensitive GIST cell lines and improved efficacy in patient-derived GIST xenografts. In addition, inhibition of MAPK signaling by imatinib was not sustained in GIST cells. An ERK rebound occurred through activation of FGF signaling, and was repressed by FGFR1 inhibition. Downregulation of Sprouty proteins played a role in the imatinib-induced feedback activation of FGF signaling in GIST cells. SIGNIFICANCE: We here show that FGFR-mediated reactivation of the MAPK pathway attenuates the antiproliferation effects of imatinib in GISTs. The imatinib-induced ERK rebound can be repressed by the FGFR inhibitor BGJ398, and combined KIT and FGFR inhibition leads to increased efficacy in vitro and in patient-derived xenografts.

Studying clonal dynamics in response to cancer therapy using high-complexity barcoding.

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.

Pharmacological and genomic profiling identifies NF-κB-targeted treatment strategies for mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.

An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide).

UNLABELLED: Castration-resistant prostate cancer (CRPC) is the most aggressive, incurable form of prostate cancer. MDV3100 (enzalutamide), an antagonist of the androgen receptor (AR), was approved for clinical use in men with metastatic CRPC. Although this compound showed clinical efficacy, many initial responders later developed resistance. To uncover relevant resistant mechanisms, we developed a model of spontaneous resistance to MDV3100 in LNCaP prostate cancer cells. Detailed characterization revealed that emergence of an F876L mutation in AR correlated with blunted AR response to MDV3100 and sustained proliferation during treatment. Functional studies confirmed that AR(F876L) confers an antagonist-to-agonist switch that drives phenotypic resistance. Finally, treatment with distinct antiandrogens or cyclin-dependent kinase (CDK)4/6 inhibitors effectively antagonized AR(F876L) function. Together, these findings suggest that emergence of F876L may (i) serve as a novel biomarker for prediction of drug sensitivity, (ii) predict a "withdrawal" response to MDV3100, and (iii) be suitably targeted with other antiandrogens or CDK4/6 inhibitors. SIGNIFICANCE: We uncovered an F876L agonist-switch mutation in AR that confers genetic and phenotypic resistance to the antiandrogen drug MDV3100. On the basis of this fi nding, we propose new therapeutic strategies to treat patients with prostate cancer presenting with this AR mutation.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development.

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.