Mechanisms Of Synergistic Anti-Leukemic Interactions Between Mk-1775and Panobinostat In Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is the most common form of acute leukemia andhas a guarded prognosis, with overall survival rates of25%and65%in the adult andpediatric populations, respectively. Therefore, there is an urgent need of novel andmore effective therapies for treating this deadly disease.Standard treatment for AML consists primarily of a combination of cytarabine(cytosine arabinoside, Ara-C) and anthracyclines (e.g. daunorubicin, DNR). Thesedrugs are thought to act by targeting DNA in dividing cells, leading to DNA damage.The DNA damage triggers activation of the cell cycle checkpoints, leading to cellcycle arrest, DNA damage repair, and cell survival. Therefore, activation of the cellcycle checkpoints remains a possible mechanism of drug resistance leading totreatment failure for AML.Checkpoint kinase1(CHK1) plays an important role in replication initiation andfork stability, homologous recombination repair, progression of the cell cycle, and theS and G2/M cell cycle checkpoints. In response to DNA damage or replication stress,CHK1phosphorylates CDC25phosphatases, inhibiting activation of CDK1/CDK2and arresting cell cycle progression allowing for DNA repair and cell survival.Another important checkpoint kinase is Wee1, whose primary function is inhibitoryphosphorylation of CDK1and CDK2on Tyr-15(Y15), which prevents cell cycleprogression. Thus, targeting these checkpoint kinases would interfere with the DNAdamage response, allowing for accumulation of irreparable DNA damage andeventually leading to cell death.Histone deacetylases (HDAC) inhibitors (HDACIs) have demonstrated anticanceractivity in numerous malignancies including AML. They have been demonstrated to induce cell cycle arrest, differentiation, and apoptosis in cancer cells, but less so innormal cells. Though their single-agent efficacy in the clinic has been modest, thereare many clinical trials investigating combination therapies (NCT01242774,NCT01742793, NCT02061449, and NCT02145715, clinicaltrials.gov). Previously, wedemonstrated that panobinostat, a pan-HDACI which has been recently approved bythe US FDA for the treatment of multiple myeloma, downregulates the CHK1pathway in AML cells. Given the critical roles CHK1and Wee1play in the DNAdamage response, we hypothesized that the combination of panobinostat andMK-1775would result in synergistic anti-leukemic activity in AML cells.To test this possibility, we first tested the anti-leukemic activities and theunderlying molecular mechanism of MK-1775, the first-in-class Wee1selectiveinhibitor, in both AML cell lines and diagnositic blast samples derived from AMLpatients either at intital diagnosis or at relapse. We found that newly diagnosed andrelapsed patient samples were equally sensitive to MK-1775. We also found thatAra-C-resistant HL-60cells were significantly more sensitive to MK-1775than theparental cells, further supporting the use of MK-1775for treating relapse AML. Inaddition, patient samples harboring t(15;17) translocation were significantly moresensitive to MK-1775than non-t(15;17) samples. MK-1775induced apoptosis in bothAML cell lines and diagnostic blast samples, accompanied by decreasedphosphorylation of CDK1and CDK2on Tyr-15and increased DNA double-strandbreaks (DSBs). Time-course experiments, using AML cell lines, revealed atime-dependent increase in DNA DSBs, activation of CHK1and subsequent apoptosisfollowing MK-1775treatment, which could be attenuated by a CDK1/2inhibitor,Roscovitine. Simultaneous inhibition of CHK1and Wee1resulted in synergisticanti-leukemic activity in both AML cell lines and primary patient samples ex vivo.We then determined the level and extent of anti-leukemic interactions betweenMK-1775and panobinostat in both AML cell lines and primary patient samples. Wefound that panobinostat had equal anti-leukemic activities in primary AML blastsderived from patients either at initial diagnosis or at relapse. We also demonstratedthat panobinostat and MK-1775synergistically induced proliferation arrest and celldeath. Interestingly, treatment with panobinostat alone or in combination withMK-1775resulted in decreased Wee1protein levels as well as downregulation of the CHK1pathway. Lentiviral shRNA knockdown of CHK1significantly sensitized AMLcells to MK-1775treatment, while knockdown of Wee1significantly enhanced bothMK-1775-and panobinostat-induced cell death.In summary, our study provides compelling evidence that CHK1plays a criticalrole in the anti-leukemic activity of MK-1775and highlights a possible mechanism ofresistance to MK-1775. In addition, our study strongly supports the use of MK-1775to treat both newly diagnosed and relapsed AML, especially cases with t(15;17)translocation. Finally, our results demonstrate that panobinostat synergizes withMK-1775in AML cells, at least in part through downregulation of CHK1and/orWee1, providing compelling evidence for the clinical development of the combinationtreatment in AML.