Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

RKIP inhibits NF-kappaB in cancer cells by regulating upstream signaling components of the IkappaB kinase complex.

RKIP was first identified as an inhibitor of the Raf-MEK-ERK signaling pathway. RKIP was also found to play an important role in the NF-kappaB pathway. Genetic and biochemical studies demonstrated that RKIP functioned as a scaffold protein facilitating the phosphorylation of IkappaB by upstream kinases. However, contrary to what one would expect of a scaffold protein, our results show that RKIP has an overall inhibitory effect on the NF-kappaB transcriptional activities. Since NF-kappaB target gene expression is subject to negative regulation involving the optimal induction of negative regulators, our data support a hypothesis that RKIP inhibits NF-kappaB activity via the auto-regulatory feedback loop by rapidly inducing the expression and synthesis of inhibitors of NF-kappaB activation.

The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.