Background: CDK12 mutations occur in ovarian cancer. Results: These mutations impaired CDK12 kinase activity. Additionally, disabling CDK12 in ovarian cancer cells reduced BRCA1 levels and disrupted homologous recombination repair. Conclusion: CDK12 mutations that impair kinase activity likely disrupt homologous recombination. Significance: Defects in homologous recombination caused by CDK12 mutations may predict sensitivity to chemotherapy agents, including poly(ADP-ribose) polymerase inhibitors.

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Replication stress and DNA damage activate the ATR-CHK1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and CHK1 kinases in ovarian cancer cells using genetic and pharmacological inhibitors of in combination with cisplatin, topotecan, gemcitabine and the poly(ADP-ribose)-polymerase (PARP) inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNAi-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan and gemcitabine each activated CHK1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 or the CHK1 inhibitor MK-8776 blocked ATR-mediated CHK1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-CHK1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. Additionally, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and CHK1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents, and that CHK1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-CHK1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, ps6kk, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable kck-derived mRNA transcripts, protein, or PTK activity. The p56kk-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56kk activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56kk-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56kk-deficient cell line displayed a profound reduction in T-cell antigen receptordependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56kk-deficient cells were completely reversed by transfection of these cells with a wild-type kk expression vector. These results indicate that p56kk expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses.Antigenic or mitogenic stimulation of quiescent (GO-phase)

, MEK, but did not inhibit activation of Raf, the canonical upstream activator of MEK. These results suggest that a novel wortmanninsensitive activation pathway regulates MEK and MAP kinase in IL-2-stimulated T lymphocytes.The interaction of a resting T cell with appropriately presented antigen initiates cell cycle entry (G 0 -to G 1 -phase transition) and the expression of high-affinity interleukin-2 (IL-2) receptors (IL-2R). The binding of IL-2 to the IL-2R then elicits G 1 -to S-phase progression and commits the cell to traverse the remainder of the cell cycle. The high-affinity IL-2R is minimally composed of an IL-2R␣ (p55), IL-2R␤ (p70), and ␥ c (p64) subunit (57). However, heterodimerization of the IL-2R␤ and ␥ c intracellular domains is sufficient to trigger the mitogenic response (42,44).Although the IL-2R subunits exhibit no intrinsic protein tyrosine kinase (PTK) activity, IL-2-induced PTK activation is requisite for proliferation (47). The IL-2R␤ chain is physically and functionally coupled to the Src-family PTKs, Lck (20,21), Fyn (25,26,36), and Lyn (59). Additionally, the newly discovered JAK family PTKs, JAK1 and JAK3, are also activated by IL-2 (22, 65). Ligand-induced PTK activation elicits the downstream activation of multiple signaling components, one of which is the well-defined Ras/MAP kinase pathway. The Ras/ MAP kinase pathway comprises a set of ubiquitous and highly conserved molecules that orchestrate the delivery of signals from the cell membrane to the nucleus (reviewed in reference 34). IL-2R ligation appears to regulate Ras via the tyrosine phosphorylation of the adaptor protein, Shc (8,50,70). Tyrosine-phosphorylated Shc then associates with the Src homology 2 (SH2) domain of the adaptor protein, Grb2 (2, 70), which is constitutively bound to the mammalian homolog of the Ras guanine-nucleotide exchange protein, Son of Sevenless (SOS) (18, 31). Complex formation results in the SOS-catalyzed exchange of GDP for GTP on Ras, with the concomitant activation of Ras (17,54). Activated Ras interacts with the regulatory N terminus of the serine-threonine kinase, Raf (38,62,64,68), localizing Raf to the membrane and providing a requisite signal for Raf activation (30, 55). Activated Raf then initiates a protein kinase cascade by phosphorylating and activating MEK (33), which, in turn, phosphorylates and activates MAP kinase (14,33,43).The studies cited above define a linear signal transduction pathway leading to MAP kinase activation. However, recent data suggest that other signaling events also regulate the activity of this signaling cascade (7,28,67,69). In addition to activating the Ras pathway, IL-2 also activates phosphatidylinositol 3-kinase (PI3-K) (4, 35, 51) via a Src family kinasedependent mechanism (25). This heterodimeric lipid kinase comprises an 85-kDa regulatory subunit and a 110-kDa catalytic subunit, which phosphorylate phosphatidylinositol at the D-3 hydroxyl of the inositol ring (reviewed in reference 15). PI3-K activation correlates closely with tyrosine kinase growth facto...