Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition.

BACKGROUND: Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models. METHODS: Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively. RESULTS: In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model. CONCLUSIONS: These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.

Preclinical evaluation of the WEE1 inhibitor MK-1775 as single-agent anticancer therapy.

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.

Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1.

Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase-arrested cells directly into mitosis without completing DNA synthesis, resulting in highly abnormal mitoses characterized by dispersed chromosomes and disorganized bipolar spindles, ultimately resulting in mitotic exit with gross micronuclei formation and apoptosis. This mechanism of cell death is shared by CHK1 inhibitors, and combined WEE1 and CHK1 inhibition forces mitotic entry from S-phase in the absence of chemotherapy. We show that p53/p21 inactivation combined with high expression of mitotic cyclins and EZH2 predispose to mitotic entry during S-phase with cells reliant on WEE1 to prevent premature cyclin-dependent kinase (CDK)1 activation. These features are characteristic of aggressive breast, and other, cancers for which WEE1 inhibitor combinations represent a promising targeted therapy.

PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.