Targeting CHK1 For The Treatment Of Acute Myeloid Leukemia

Acute myeloid leukemia remains a devastating hematologic malignancy.The standard treatment strategy for most AML patients is ‘7+3 therapy’(7 days of cytarabine(ara-C)treatment followed by 3 days of anthracycline [e.g.,daunorubicin(DNR)treatment],which has been used for over 40 years.However,most AML patients relapse.Therefore,more effective therapies are urgently needed to overcome resistance to chemotherapy and improve the overall survival rate of AML patients.Many studies suggest that one of the promising strategies to overcome chemotherapeutic resistance is by targeting the DNA damage response(DDR).Checkpoint Kinase 1(CHK1)plays a crucial role in the DDR and its inhibition can affect replication initiation and fork stability.In addition,CHK1 inhibition leads to dephosphorylation of CDK1/CDK2 on Tyr-15 residue(Y15),allowing CDK1/CDK2 to remain active.Overactivaiton of CDK1/CDK2 causes abrogation of the S and G2/M cell cycle checkpoints,denying the cells adequate time to repair DNA damage,resulting in increased DNA damage and ultimately induces cell death.LY2603618 is a novel CHK1-selective inhibitor.It has superior selectivity compared to the first generation of CHK1 inhibitors,but the underlying molecular mechanism of LY2603618 has not been fully understood.To elucidate the underlying molecular mechanism,we tested the anti-leukemic activities of LY2603618 in both AML cell lines and diagnostic blast samples derived from AML patients either at initial diagnosis or at relapse by MTT assay.We found that newly diagnosed and relapsed patient samples had similar sensitivities to LY2603618.We also demonstrated that LY2603618 treatment could induce DNA damage which was partially dependent on CDK activity.It has been reported that DNA damage can cause downregulation of anti-apoptotic protein Mcl-1.Accordingly,we found that LY2603618 treatment decreased expression of Mcl-1 protein and that this decrease contributed to LY2603618-induced apoptosis.ABT-199 is a Bcl-2 selective inhibitor which has shown promising anti-leukemic activity.However,we have previously demonstrated that Mcl-1 plays a role in intrinsic resistance to ABT-199.Thus,we hypothesized that the combination of LY2603618 and ABT-199 would overcome this mechanism of resistance to ABT-199 and significantly enhance ABT-199’s anti-leukemic activity in AML cells.To test this possibility,we treated AML cells and primary AML patient samples with the combination of LY2603618 and ABT-199 and found that LY2603618 enhanced the anti-leukemic activity of ABT-199 by abrogating the increase of Mcl-1 induced by ABT-199 treatment in AML cells.Interestingly,we also found that ABT-199 treatment enhanced DNA damage induced by LY2603618 and further induced AML cell death.These results support further clinical development of the combination of LY2603618 and ABT-199 for the treatment of AMLRelapse remains a major hindrance to successful treatment of AML in the clinic.So far,there are no effective treatment strategies for relapsed AML patients.Despite initial response,most AML patients relapse after chemotherapy treatment and are often resistant to further chemotherapy.Based on this,we speculated that chemotherapy resistant AML cells become more reliant on the S and G2/M cell cycle checkpoints to allow them enough time to repair DNA damage and survive chemotherapy treatment.However,this potential dependence on the S and G2/M cell cycle checkpoints may also make them more susceptible to S and G2/M checkpoint inhibition.To test this possibility,we established two Ara-C resistant AML cell lines,HL-60/Ara-C and CMK/Ara-C,and then determined their sensitivity to CHK1(LY2603618 and MK8776),ATR(AZ20 and AZD6738)and WEE1(MK1775)inhibitors.Interestingly,we found that compared to the parental HL-60 cells,HL-60/Ara-C cells were significantly more sensitive to the cell cycle checkpoint inhibitors mentioned above.CMK/Ara-C cells were significantly sensitive to ATR inhibitor AZD6738 and tended to be more sensitive to other cell cycle checkpoint inhibitors compared to CMK parental cells.Additionally,LY2603618 in combination with AZD6738 or MK1775 resulted in synergistic antileukemic activities in both Ara-C resistant AML cell lines and did not have toxicity to normal cells.In summary,our study provides compelling evidence that CHK1 plays a critical role in the treatment of AML.We elucidated a possible mechanism of LY2603618 treatment in AML cells and demonstrated that LY2603618 can enhance ABT-199 anti-leukemic activity by inhibiting Mcl-1.Finally,we demonstrated that combined LY2603618 and AZD6738 or MK1775 results in synergistic antitumor activity in Ara-C resistant AML cells.Taken together,these results provide support for the clinical development of CHK1 inhibitors alone and in combination treatments for the treatment of AML.