New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

T-cell acute lymphocytic leukemia (T-ALL) affects approximately 1,500 people per year in the United States, many of them children. The overall survival rate of children with T-ALL is greater than 80%. However, patients in a newly identified subtype called early T-cell progenitor acute lymphocytic leukemia (ETP-ALL), have a survival rate of only 19%. We have used the Sleeping Beauty (SB) transposon/transposase system in mice to model the T-ALL subtypes and identify cancer-causing mutations in the diseases. We have also developed three strains of NOTCH1 transgenic mice. NOTCH1 is a gene that is mutated in over 60% of cases of T-ALL, and these NOTCH1 transgenic mice could be used to better understand the role NOTCH1 plays in T-cell transformation. In order to model T-ALL subtypes we crossed SB mice to Vav-iCre mice, Lck-Cre mice, and CD4-Cre mice. This causes activation of SB in hematopoietic stem cells (HSCs), double negative (DN) thymocytes, and double positive (DP) T-cells, respectively. The Vav/SB tumors were characterized by mutations in Notch1, Ikzf1, and Rasgrp1. The predominant mutations found in the Lck/SB and CD4/SB models were Stat5b, Myc, Gfi1, Whsc1, and Jak1. Microarray was performed on a subset of samples. It was found that the CD4/SB tumors had expression profiles very similar to human ETP-ALL samples, and the Vav/SB tumors had expression profiles very similar to typical T-ALL samples. This data indicates that the cell-of-origin for ETP-ALL may not be an early T-cell progenitor, but instead may be a double positive or single positive T-cell. It also indicates that the cell-of-origin for typical T-ALL may be a stem/progenitor cell of the T-lineage. In order to better study the oncogenic potential of mutant NOTCH1 in T- ALL, we produced three NOTCH1 transgenic mouse strains that mimic the most common NOTCH1 mutations found in human T-ALL patients. These mutations are found in two distinct regions of the gene, the heterodimerization (HD) domain and the PEST domain and can occur alone or in combination. The strains also contain a lox-stop-lox (LSL) cassette in the first exon of NOTCH1, making expression Cre-dependent. Preliminary results from NOTCH1-HD-PEST (NHDP) transgenic mice indicate that, when crossed to Lck-Cre mice, offspring do not develop T- cell lymphoma. However, when NHDP/Lck-Cre mice are crossed to SB mice, the mutant NOTCH1 transgene accelerates a SB-induced model of T-cell lymphoma.