Drugging DNA Repair to Target T-ALL Cells

Abstract

Despite recent progress in treatment, prognostication for T-cell acute lymphoblastic leukemia (T-ALL) patients, including those with early T-cell precursor ALL (ETP-ALL) is rather poor. Therefore, new therapies are needed to extend the complete remission time/cure and/or to treat refractory T-ALL patients. T-ALL cells accumulate potentially lethal “spontaneous” and drug-induced DNA double-strand breaks (DSBs). Thus, T-ALL cells may be “addicted” to DSB repair mechanisms to survive and targeting these pathways should sensitize them to lethal effect of DSBs. In proliferating cells DSBs are usually repaired by homologous recombination (HR) repair. HR works predominantly on broken replication forks and usually depends on BRCA1, BRCA2, PALB2, RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), and RAD51 (BRCA pathway). However, in BRCA deficient cells displaying low expression of at least one of the proteins from the pathway, PARP1-LIG3 -mediated alternative non-homologous end-joining (Alt-NHEJ) and/or RAD52-RAD51-dependent alternative HR mechanism protect cells from lethal effect of DSBs. Therefore, PARP1 and/or RAD52 are attractive targets to eliminate BRCA-deficient leukemia cells. Although inactivating mutations in genes involved in HR are extremely rare in T-ALL, we show here that microarray gene expression profiling of T-ALLs displayed wide range of expression of mRNAs encoding the proteins in BRCA pathway suggesting that numerous leukemias from individual patients were BRCA deficient. In particular, lower expression of BRCA genes (BRCA1, BRCA2, XRCC2, RAD51B, RAD51C, RAD51) have been detected in ETP-ALLs. These data implicate malfunctioning BRCA pathway in a cohort of T-ALLs, especially in ETP-ALLs. Conversely, an average expression of PARP1 and LIG3 mRNAs seemed not to be affected, and RAD52 mRNA was even overexpressed in ETP-ALLs when compared to T-ALLs. Next, we tested the sensitivity of primary leukemia xenograft (PLX) CD3+ cells from T-ALL/ETP-ALL patients and of normal T cells to DNA repair inhibitors. All 3 samples of ETP-ALL cells were more sensitive to PARP1 inhibitor olaparib and RAD52 inhibitor 6(OH)-DL-dopa when compared to normal pan-T cells. T-ALL PLXs displayed differences in sensitivity to PARP1 and RAD52 inhibitors; two samples appeared sensitive whereas three seemed resistant. In addition, cytotoxic drug Ara-C combined with PARP1 or RAD52 inhibitors exerted stronger anti-leukemia activity when compared to individual treatments in PARP1/RAD52 inhibitor-sensitive leukemia cells. To determine if the differences in sensitivity to PARP1 and RAD52 inhibitors were associated with deficiencies in HR activity, sensitive and resistant leukemia cells as well as normal pan-T cells were transfected with the plasmid containing HR reporter cassette. HR activity was determined by restoration of a functional GFP gene and detection of GFP+ cells. T-ALL/ETP-ALL cells which were sensitive to PARP1 and RAD52 inhibitors displayed diminished HR activity in comparison to normal pan-T cells and T-ALL cells resistant to the inhibitors. Moreover, BRCA1 and RAD51 nuclear foci formation was inhibited in leukemia cells which displayed reduced HR activity and increased sensitivity to PARP1 and RAD52 inhibitors. In summary, our results suggest that majority of ETP-ALLs and a subset of T-ALLs display “BRCAness” phenotype, which predisposes them to be sensitive to PARP1 and RAD52 inhibitors. While RAD52 inhibitors await clinical development, PARP1 inhibitors such as Lynparza (olaparib), Rubraca (rucaparib) and Zeluja (niraparib) have been FDA approved to treat breast and/or ovarian cancers carrying BRCA1/2 mutations. We postulate that these inhibitors may be used to improve therapeutic effect of standard cytotoxic drugs in pre-selected patients with “BRCAness” T-ALLs/ETP-ALLs. DisclosuresMullighan:Loxo Oncology: Research Funding; Amgen: Consultancy.