An Inhibitor of the Pleckstrin Homology Domain of CNK1 Selectively Blocks the Growth of Mutant KRAS Cells and Tumors.

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain-containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS-driven cancer. SIGNIFICANCE: These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.

Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target.

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.

A new HIF-1α/RANTES-driven pathway to hepatocellular carcinoma mediated by germline haploinsufficiency of SART1/HAF in mice.

UNLABELLED: The hypoxia-inducible factor (HIF), HIF-1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating nonhypoxic induction of HIF-1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen-independent ubiquitin ligase for HIF-1α: hypoxia-associated factor (HAF; encoded by SART1). SART1(-/-) mice were embryonic lethal, whereas male SART1(+/-) mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1(+/-) mice showed significant up-regulation of HIF-1α in circulating and liver-infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1(+/-) mice produced increased levels of the HIF-1-dependent chemokine, regulated on activation, normal T-cell expressed and secreted (RANTES), compared to wild type. This was associated with increased liver-neutrophilic infiltration, whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration and attenuated HCC tumor initiation/growth in SART1(+/-) mice. CONCLUSION: This work establishes a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

Modulating microtubule stability enhances the cytotoxic response of cancer cells to Paclitaxel.

The extracellular matrix protein TGFBI enhances the cytotoxic response of cancer cells to paclitaxel by affecting integrin signals that stabilize microtubules. Extending the implications of this knowledge, we tested the more general hypothesis that cancer cell signals which increase microtubule stability before exposure to paclitaxel may increase its ability to stabilize microtubules and thereby enhance its cytotoxicity. Toward this end, we carried out an siRNA screen to evaluate how genetic depletion affected microtubule stabilization, cell viability, and apoptosis. High content microscopic analysis was carried out in the absence or presence of paclitaxel. Kinase knockdowns that stabilized microtubules strongly enhanced the effects of paclitaxel treatment. Conversely, kinase knockdowns that enhanced paclitaxel-mediated cytotoxicity sensitized cells to microtubule stabilization by paclitaxel. The siRNA screen identified several genes that have not been linked previously to microtubule regulation or paclitaxel response. Gene shaving and Bayesian resampling used to classify these genes suggested three pathways of paclitaxel-induced cell death related to apoptosis and microtubule stability, apoptosis alone, or neither process. Our results offer a functional classification of the genetic basis for paclitaxel sensitivity and they support the hypothesis that stabilizing microtubules prior to therapy could enhance antitumor responses to paclitaxel treatment.

Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory.

In recent years high throughput screening operations have become a critical application in functional and translational research. Although a seemingly unmanageable amount of data is generated by these high-throughput, large-scale techniques, through careful planning, an effective Laboratory Information Management System (LIMS) can be developed and implemented in order to streamline all phases of a workflow. Just as important as data mining and analysis procedures at the end of complex processes is the tracking of individual steps of applications that generate such data. Ultimately, the use of a customized LIMS will enable users to extract meaningful results from large datasets while trusting the robustness of their assays. To illustrate the design of a custom LIMS, this practical example is provided to highlight the important aspects of the design of a LIMS to effectively modulate all aspects of an siRNA screening service. This system incorporates inventory management, control of workflow, data handling and interaction with investigators, statisticians and administrators. All these modules are regulated in a synchronous manner within the LIMS.

SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer.

Regulators of mitosis have been successfully targeted to enhance response to taxane chemotherapy. Here, we show that the salt inducible kinase 2 (SIK2) localizes at the centrosome, plays a key role in the initiation of mitosis, and regulates the localization of the centrosome linker protein, C-Nap1, through S2392 phosphorylation. Interference with the known SIK2 inhibitor PKA induced SIK2-dependent centrosome splitting in interphase while SIK2 depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. We believe these data identify SIK2 as a plausible target for therapy in ovarian cancers.