Cotargeting CDK4/6 and BRD4 Promotes Senescence and Ferroptosis Sensitivity in Cancer.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell-cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Cotargeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors. SIGNIFICANCE: The combination of cytostatic CDK4/6 and BRD4 inhibitors induces senescent cancer cells that are primed for activation of ferroptotic cell death by targeting GPX4, providing an effective strategy for treating cancer.

MOB3A Bypasses BRAF and RAS Oncogene-Induced Senescence by Engaging the Hippo Pathway.

Oncogenic activation of the RTK-RAS-RAF-MEK-ERK pathway occurs in approximately 25% of all human cancers, yet activated RAS, BRAF, or MEK expression in primary cells leads to a prolonged and predominantly irreversible cell-cycle arrest termed oncogene-induced senescence (OIS). OIS acts as an intrinsic tumor suppressor mechanism, serving as a barrier to tumor progression. Screening a library of activated kinases and kinase-regulatory proteins we identified MOB3A, a Mps-one binder coactivator (MOB) protein family member, whose constitutive expression permits proliferation and suppresses senescence in response to oncogenic RAS and BRAF signals. MOB3A is one of seven human MOB genes, which are highly conserved from yeast to human and that function to activate the Hippo pathway kinases (MST/LATS) or NDR kinases through direct association. Here we show that within the MOB family of genes MOB3A and C are unique in their ability to allow primary cell proliferation in the face of sustained oncogene signaling. Unlike the canonical MOB1A/B proteins, MOB3A inhibits Hippo/MST/LATS signaling and constitutive MOB3A membrane localization phenocopies OIS bypass seen with elevated YAP expression. Moreover, inhibition of MOB3 family member expression results in decreased proliferation and tumor growth of cancer cell lines. Together these data identify MOB3A's role in bypass of oncogene induced senescence and its role as a Hippo pathway inhibitor. IMPLICATIONS: These results suggest that MOB3 targeting to re-engage the Hippo pathway, or direct targeting of YAP/TAZ, may be viable therapeutic strategies potential for RAS-pathway driven tumours.

Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence.

Cellular senescence is characterized by a prolonged and predominantly irreversible cell-cycle arrest state, which is linked to loss of tissue function and aging in mammals. Moreover, in response to aberrant oncogenic signals such as those from oncogenic RAS or BRAF, senescence functions as an intrinsic tumor suppressor mechanism restraining tumor progression. In addition to this durable proliferative block, senescent cells adopt altered morphologies, transcriptional profiles, and metabolism, while often possessing unusual heterochromatin formation termed senescence-associated heterochromatic foci. To uncover genes that are required to permit proliferation in the face of sustained oncogene signaling, we conducted an shRNA-based genetic screen in primary cells expressing inducible BRAF. Here we show that depletion of a known glycolysis regulator, islet amylin polypeptide (IAPP also known as amylin), prevents RAS and BRAF oncogene-induced senescence (OIS) in human cells. Importantly, depletion of IAPP resulted in changes of the cells' metabolome and this metabolic reprogramming was associated with widespread alterations in chromatin modifications compared with senescent cells. Conversely, exogenous treatment of IAPP-depleted cells with amylin restored OIS. Together, our results demonstrate that the metabolic regulator IAPP is important regulator of OIS. Moreover, they suggest that IAPP analog treatment or activation of IAPP signaling in RAS/BRAF mutant tumors may have therapeutic potential through senescence induction. IMPLICATIONS: These findings demonstrate that IAPP is a novel metabolic regulator of oncogene-induced senescence and use of IAPP analogs may be therapeutically effective to restore growth arrest to BRAF and/or RAS mutant cancers.

A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.