Abstract
New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies.
To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156.
Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 < 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (>9 folds, p<0.01), activated JAK/STAT pathway and lower levels of MYC. Sensitive cells (H-SB2 and DU.528), had an immature or ETP-ALL phenotype (CD4-/CD8- and TdT-). Insensitive CUTLL1, SUP-T1, and HPB-ALL (XTT assay: IC50 > 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells.
While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI < 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rɣ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p<0.05), and reduced the number of leukemic cells (hCD45 staining) in the peripheral blood and bone marrow. This dual therapy significantly prolonged the survival (p<0.005) of the treated mice. This outcome was obtained at one-tenth of normal dose of Ponatinib used in animals, suggesting that in the humans the side effects induced by this agent could be avoided by using this combination therapy.
Taken together, these findings highlight the important role for PIM protein kinase activity in T-ALL and especially in the ETP-ALL, phenotype. Tumor xenograft experiments provide strong preclinical rationale for a novel treatment strategy of combining PIM and tyrosine kinase inhibitors for treatment of patients with PIM overexpressing T-ALL.
Figure 1 Combination treatment of AZD1208 and Ponatinib in H-SB2-luc xenograft model, significantly suppressed leukemia burden as compared with vehicle-treated group. Figure 1. Combination treatment of AZD1208 and Ponatinib in H-SB2-luc xenograft model, significantly suppressed leukemia burden as compared with vehicle-treated group.
Disclosures
No relevant conflicts of interest to declare.
mTOR kinase inhibitor sensitizes T-cell lymphoblastic leukemia for chemotherapy-induced DNA damage via suppressing FANCD2 expression
