Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation.

Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely quantify cell-cycle effects and diverse cell states orchestrated by specific CDKs. While pairwise disruption of CDK4/6 is synthetic-lethal, only CDK6 is required for normal cell-cycle progression and transcriptional activation. Multiple CDKs (CDK1/7/9/12) are synthetic-lethal in combination with PRMT5, independent of cell-cycle control. In-depth analysis of mRNA expression and splicing patterns provides multiple lines of evidence that the CDK-PRMT5 dependency is due to aberrant transcriptional regulation resulting in premature termination. These inter-dependencies translate to drug-drug synergies, with therapeutic implications in cancer and other diseases

Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation

Abstract Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely quantify cell-cycle effects and diverse cell states orchestrated by specific CDKs. While pairwise disruption of CDK4/6 is synthetic-lethal, only CDK6 is required for normal cell-cycle progression and transcriptional activation. Multiple CDKs (CDK1/7/9/12) are synthetic-lethal in combination with PRMT5, independent of cell-cycle control. In-depth analysis of mRNA expression and splicing patterns provides multiple lines of evidence that the CDK-PRMT5 dependency is due to aberrant transcriptional regulation resulting in premature termination. These inter-dependencies translate to drug–drug synergies, with therapeutic implications in cancer and other diseases

A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation.

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA

Pomalidomide and lenalidomide regulate erythropoiesis and fetal hemoglobin production in human CD34+ cells

Sickle-cell disease (SCD) and β thalassemia constitute worldwide public health problems. New therapies, including hydroxyurea, have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve current treatment. Lenalidomide and pomalidomide are members of a class of immunomodulators used as anticancer agents. Because clinical trials have demonstrated that lenalidomide reduces or eliminates the need for transfusions in some patients with disrupted blood cell production, we investigated the effects of lenalidomide and pomalidomide on erythropoiesis and hemoglobin synthesis. We used an in vitro erythropoiesis model derived from human CD34+ progenitor cells from normal and SCD donors. We found that both compounds slowed erythroid maturation, increased proliferation of immature erythroid cells, and regulated hemoglobin transcription, resulting in potent induction of HbF without the cytotoxicity associated with other HbF inducers. When combined with hydroxyurea, pomalidomide and, to a lesser extent, lenalidomide were found to have synergistic effects on HbF upregulation. Our results elucidate what we believe to be a new mechanism of action of pomalidomide and lenalidomide and support the hypothesis that pomalidomide, used alone or in combination with hydroxyurea, may improve erythropoiesis and increase the ratio of fetal to adult hemoglobin. These findings support the evaluation of pomalidomide as an innovative new therapy for β-hemoglobinopathies

CC-509 inhibits paw swelling in rat collagen-induced arthritis model.

<p>Change in paw swelling from study day 11 to day 18 in non-immunized rats (black line) or collagen-immunized rats dosed with vehicle (blue line), indomethacin (light green line), or CC-509 at 15 mg/kg q.d. (dark green line), 15 mg/kg b.i.d. (orange line), or 25 mg/kg q.d. (red line). Data points are mean ± SEM. Statistical significance at **p<0.01, ***p<0.001, and ****p<0.0001.</p

Biochemical characterization of CC-509.

<p>(A) Chemical structure of CC-509 with IUPAC name of [3-(2H-3,4,5,6-tetrahydropyran-4-ylamino)(1H-indazol-6-yl)][5-(2H-3,4,5,6-tetrahydropyran-4-ylmethyl)(2H-1,2,4-triazolo[1,5-a]pyridin-2-yl)]amine. (B) Representative IC₅₀ curve (top panel) and K_i determination (bottom panel) for CC-509 tested against recombinant Syk enzyme. (C) Michaelis-Menten (left panel) and Lineweaver-Burk (right panel) plots for Syk enzyme tested against increasing amounts of substrate and concentrations of CC-509 (shown in nM). (D) Crystallographic diagram of Syk ATP pocket when bound with CC-509. Key residue alanine 451 and hydrogen bonds are shown.</p

CC-509 inhibits proinflammatory chemokine production in rat collagen-induced arthritis model.

<p>Proinflammatory chemokine (clockwise from upper right: MIP-1α, KC, MCP-1, RANTES) levels on day 18 in paw tissue from non-immunized rats (white bars) or collagen-immunized rats dosed with vehicle (black bars), indomethacin (Indo, blue bars), or CC-509 at 15 mg/kg q.d. (dark green bars), 15 mg/kg b.i.d. (light blue bars), or 25 mg/kg q.d. (light green bars). Data points are mean ± SEM. Statistical significance at * = p<0.05, **p<0.01, ***p<0.001.</p

FcR-dependent functional activity in primary human immune cells is inhibited by CC-509.

<p>When stimulated through the indicated FcR, CC-509 inhibits functional activity in (A) primary human macrophage, (B) primary human B-cells and (C) the LAD2 human mast cell line. (D) <i>Left panel</i>: CC-509 inhibits CD63 upregulation on basophils stimulated through indicated FcR in human whole blood. <i>Right panel</i>: Representative flow-cytometry plot showing the number of events and CD63 mean fluorescence intensity (MFI) on unstimulated (brown trace) or stimulated basophils in the presence of 0.1 μM (blue trace), 1.0 μM (green trace), or 10.0 μM (red trace) CC-509. IC₅₀ values (μM) are indicated in upper-right portion of each panel. All data expressed as percent of DMSO-treated control, mean ± SD.</p