The TGF-β signaling mediator SMAD4 is frequently mutated or deleted in colorectal and pancreatic cancers. SMAD4 acts as a tumor suppressor and its loss is associated with poorer patient outcomes. The purpose of this study was to find synthetic lethal interactions with SMAD4 deficiency in order to find novel therapeutic strategies for the treatment of patients with SMAD4-deficient colorectal or pancreatic cancers. Using pooled lentiviral sgRNA libraries, we conducted genome-wide loss-of-function screens in Cas9-expressing colorectal and pancreatic cancer cells harboring altered or wild-type SMAD4. The small GTPase protein RAB10 was identified and validated as a susceptibility gene in SMAD4-altered colorectal and pancreatic cancer cells. Rescue assays showed that RAB10 re-introduction reversed the anti-proliferative effects of RAB10 KO in SMAD4 negative cell lines. Further investigation is necessary to shed light on the mechanism by which RAB10 inhibition decreases cell proliferation of SMAD4-negative cells.

<div>Abstract<p>The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of <i>BRAF</i>-mutant (<i>BRAF</i>^MUT) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant <i>PTEN</i> loss of function (<i>PTEN</i>^LOF) occurs in approximately 40% of <i>BRAF</i>^MUT melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of <i>PTEN</i>^LOF<i>/BRAF</i>^MUT melanoma. Large-scale compound sensitivity profiling revealed that <i>PTEN</i>^LOF melanoma cell lines were sensitive to PI3Kβ inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kβ inhibitor identified an adaptive response involving the IGF1R–PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kβ, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in <i>PTEN</i>^LOF<i>/BRAF</i>^MUT melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kβ + IGF1R, and MAPK pathway inhibitors in <i>PTEN</i>^LOF<i>/BRAF</i>^MUT melanoma patients to achieve maximal response. <i>Cancer Res; 76(2); 390–402. ©2015 AACR</i>.</p></div>

<p>Supplementary materials and methods.</p>

<p>A) <i>PTEN^LOF</i>/<i>BRAF^MUT</i> melanoma lines were treated with serial dilutions of the indicated drugs and their effect on phosphorylated and total Akt was evaluated by RPPA. B) <i>PTEN^LOF</i> calls in CCLE translate into absence of PTEN protein as evaluated by immunoblotting in a panel of <i>BRAF^MUT</i> melanoma lines using antibodies targeting the amino- and carboxy-terminus of PTEN. PI3Kβi1/2=rac-KIN-193/TGX221, panPI3Ki=GDC0941, PI3Kαi=BYL719</p>

<p>Gene_scoreMageckComparison</p>