Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy

Abstract

Key Points• Oncogenic KRAS causes upregulation of components of the alt-NHEJ pathway, thereby promoting genomic instability.• Inhibition of the alt-NHEJ pathway selectively sensitizes KRAS-mutant leukemic cells to cytotoxic agents.Activating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3a, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference-mediated knockdown of DNA ligase 3a abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations. (Blood. 2014;123(15):2355-2366

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

“…GEMA, with emphasis on gene mutation analysis, may also be applied to identify additional oncogenes that sensitize cells to PARP1 inhibitor treatment, for example TMPRSS2-ERG-positive prostate carcinomas (DNA-PKcs deficiency), EGFR mutant-positive lung carcinomas (Fanconi anemia deficiency), EWSR1-FLI1-positive Ewing's sarcomas, and KRAS mutant-positive leukemias (40)(41)(42)(43).…”

Molecular Pathways: Targeting the Dependence of Mutant RAS Cancers on the DNA Damage Response

“…A recent study also showed that mutant Ras expression confers heightened DNA repair capability through the upregulation of CUX1, which functions in BER as an ancillary factor that stimulates the activity of the OGG1 DNA glycosylase (23). Another study found that inhibition of the alt-NHEJ pathway selectively sensitized KRAS mutant leukemic cells to cytotoxic agents (24). Mutant K-Ras was shown to activate alt-NHEJ by upregulating DNA ligase III, poly (ADP-ribose) polymerase 1 (PARP1), and XRCC1.…”

INPP4B-mediated DNA repair pathway confers resistance to chemotherapy in acute myeloid leukemia

“…As is reported, ATM is a protein kinase that phosphorylates several key proteins involved in the DDR. It can be activated by its autophosphorylation at Ser1981 position [20], and it can also phosphorylate its downstreams of ATR, BRCA1, and RAD51 to activate the HR DNA repair [19,31], so we speculated that the inhibition of p-ATM, p-ATR, p-BRCA1, and p-RAD51 may be ascribed to ATM inhibition. To further identify whether INPP4B-mediated DNA repair is ATMdependent or not, we then evaluated the influences of KU55933 and Chloroquine on INPP4B knockdown-induced sensitizing effect on cytarabine treatment.…”

“…p-H2AX is a well-known maker of DNA double-strand breaks, H2AX is phosphorylated at the sites of DSBs and the level of p-H2AX has been shown to give a sensitive and accurate estimation of the number of DSBs within DNA. The presence of p-H2AX in chromatin can be detected shortly after induction of DSBs in the form of discrete nuclear foci [20]. To explore if the difference of this cell viabilities is associated with the distinction of DSBs formation, then we detected the p-H2AX expression between KG-1 and HL60 cells after cytarabine treatment, and observed p-H2AX expression is higher in HL60 than KG-1 cells.…”

“…However, cancer cells usually have DNA repair pathway deficiencies, which render cancer cells more vulnerable to genotoxic treatment but can also drive resistance if the DNA repair pathway is excessively activated [20]. Increased DNA repair contributes to drug resistance and the risk of disease relapse [21].…”