Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

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

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.

Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory Tertiary Lymphoid Organs (rTLOs). scRNAseq data and adoptive transfer of alloreactive T cells post-transplant showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required since adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8 KO recipients resulted in acceptance and not rejection. Analysis of scRNAseq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

CRISPR Screening Identifies Mechanisms of Resistance to KRASG12C and SHP2 Inhibitor Combinations in Non-Small Cell Lung Cancer.

Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non-small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments. SIGNIFICANCE: Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005.

Expressed Barcoding Enables High-Resolution Tracking of the Evolution of Drug Tolerance.

For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. SIGNIFICANCE: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.

Targeting the IL1ß Pathway for Cancer Immunotherapy Remodels the Tumor Microenvironment and Enhances Antitumor Immune Responses.

High levels of IL1ß can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1ß could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1ß blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFß treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1ß blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1ß alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1ß inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1ß blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1ß inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

CRISPR screening identifies T cell-intrinsic regulators of CD3-bispecific antibody responses.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics.

Exquisite Sensitivity to Dual BRG1/BRM ATPase Inhibitors Reveals Broad SWI/SNF Dependencies in Acute Myeloid Leukemia.

Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myelogenous leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared with other lineages. This result was striking in comparison with data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes, including MYC, a well-established target of BRG1 activity in AML. Overall, small-molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. IMPLICATIONS: Our studies reveal the breadth of SWI/SNF dependency in leukemia and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.

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.

Therapeutic Assessment of Targeting ASNS Combined with l-Asparaginase Treatment in Solid Tumors and Investigation of Resistance Mechanisms.

Asparagine deprivation by l-asparaginase (L-ASNase) is an effective therapeutic strategy in acute lymphoblastic leukemia, with resistance occurring due to upregulation of ASNS, the only human enzyme synthetizing asparagine (Annu. Rev. Biochem. 2006, 75 (1), 629-654). l-Asparaginase efficacy in solid tumors is limited by dose-related toxicities (OncoTargets and Therapy 2017, pp 1413-1422). Large-scale loss of function genetic in vitro screens identified ASNS as a cancer dependency in several solid malignancies (Cell 2017, 170 (3), 564-576.e16. Cell 2017, 170 (3), 577-592.e10). Here we evaluate the therapeutic potential of targeting ASNS in melanoma cells. While we confirm in vitro dependency on ASNS silencing, this is largely dispensable for in vivo tumor growth, even in the face of asparagine deprivation, prompting us to characterize such a resistance mechanism to devise novel therapeutic strategies. Using ex vivo quantitative proteome and transcriptome profiling, we characterize the compensatory mechanism elicited by ASNS knockout melanoma cells allowing their survival. Mechanistically, a genome-wide CRISPR screen revealed that such a resistance mechanism is elicited by a dual axis: GCN2-ATF4 aimed at restoring amino acid levels and MAPK-BCLXL to promote survival. Importantly, pharmacological inhibition of such nodes synergizes with l-asparaginase-mediated asparagine deprivation in ASNS deficient cells suggesting novel potential therapeutic combinations in melanoma.

A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy.

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.

Inhibition of MDM2 Promotes Antitumor Responses in p53 Wild-Type Cancer Cells through Their Interaction with the Immune and Stromal Microenvironment.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell-mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors. SIGNIFICANCE: This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.

DOT1L inhibition is lethal for multiple myeloma due to perturbation of the endoplasmic reticulum stress pathway.

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

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.

TGFß-blockade uncovers stromal plasticity in tumors by revealing the existence of a subset of interferon-licensed fibroblasts.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFß in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFß and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

The Discovery of SWI/SNF Chromatin Remodeling Activity as a Novel and Targetable Dependency in Uveal Melanoma.

Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no approved targeted therapies and very few effective treatments for this cancer. Although activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, few additional drug targets have been identified. Recently, studies have identified context-specific roles for the mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer lineages. Here, we find evidence that the SWI/SNF complex is essential through analysis of functional genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools and small-molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage-specific transcription factor Microphthalmia-associated Transcription Factor, suggesting that these two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the treatment of this cancer.

aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation.

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.

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.