NORMAL ABL1 IS A TUMOR SUPPRESSOR AND THERAPEUTIC TARGET IN BCR-ABL1-POSITIVE LEUKEMIAS

Abstract

BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation resulting in BCR-ABL1 kinase expression, initiating chronic myeloid leukemia in chronic phase (CML-CP). At the initial stages of CML-CP both oncogenic BCR-ABL1 kinase and normal ABL1 kinase are expressed, however, loss of ABL1 kinase expression in CML-CP can result from an interstitial deletion in the normal chromosome 9 [del(9q34)] which may be combined with the transcriptional silencing of the alternative ABL1 promoter within the translocation eventually leading to disease progression and drug resistance. We found that BCR-ABL1 Abl1-/- cells generated a CML-blast phase (BP)-like disease phenotype in NOD-SCID mice compared to the BCR-ABL1 Abl1+/+ cells. To determine the mechanisms responsible for blastic transformation of BCR-ABL1 Abl1-/- cells, we examined the role of ABL1 in proliferation, differentiation, apoptosis, genomic instability, and stemness. The presence of ABL1 inhibited proliferation in BCR-ABL1 cells as BCR-ABL1 Abl1-/- cells had higher clonogenic activity and proliferative rate compared to their wild-type counterparts. ABL1 is essential for myeloid differentiation since BCR-ABL1 Abl1-/- cells showed an immature blast phenotype when stained with Wright-Giemsa and myeloid differentiation markers Gr-1 and CD11b. ABL1 promoted apoptosis in response to genotoxic stress as revealed by reduced clonogenicity and expression of p53, phosphoserine-15 p53 and activated caspase 3 in BCR-ABL1 Abl1+/+ compared to knock-out cells. Although the absence of ABL1 did not enhance ROS and oxidative DNA damage, it appears that an impaired DNA damage response may be responsible for higher chromosome numbers and an accumulation of high numbers of chromosomal aberrations in BCR-ABL1 Abl1-/- cells. We detected an expansion of Lin-c-Kit+Sca-1+ leukemia stem cells (LSCs) in BCR-ABL1 Abl1-/- cells compared to BCR-ABL1 Abl1+/+ or non-transformed counterparts; among the LSCs, there was a higher percentage of CD34-Flt3- long-term and CD34+Flt3- short-term stem cells. These results showed that ABL1 is involved in regulating the LSC compartment in BCR-ABL1 cells. DNA microarray analysis revealed changes in mRNA levels of several genes involved in proliferation, myeloid differentiation, apoptosis, DNA damage response and `stemness' in BCR-ABL1 Abl1-/- cells in comparison to BCR-ABL1 Abl1+/+ cells. Together, these results demonstrate a critical role of ABL1 as a tumor suppressor in BCR-ABL1-induced leukemia, prolonging survival in mice by suppressing proliferation and expansion of LSC, inducing myeloid differentiation, apoptosis and DNA damage response in BCR-ABL1 cells. Loss of ABL1 was also found to contribute to Imatinib resistance in BCR-ABL1 cells. Moreover, we hypothesized that enhancement of the tumor-suppressor function of ABL1 may have a significant impact on CML treatment. A small molecule activator of ABL1 kinase, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin (DPH), have been reported to interact with the myristoyl-binding site of ABL1 and destabilize the bent conformation of the á1 helix, thereby preventing the auto-inhibitory conformation. Western blot analysis revealed partially restored activation of ABL1 kinase when Imatinib-treated cells were incubated with DPH. DPH along with Imatinib was found to inhibit viability of BCR-ABL1 Abl1+/+ cells but not BCR-ABL1 Abl1-/- cells demonstrating its ABL1-specific mode of action. DPH when used in combination with tyrosine kinase inhibitors such as Imatinib and Ponatinib inhibited growth of CML CD34+ cells, Philadelphia chromosome-positive B-Acute Lymphoblastic Leukemia (Ph+B-ALL) cells and relapsed Ph+B-ALL cells harboring T315I mutation without affecting normal counterparts. A similar inhibitory effect was observed when TEL-ABL1-expressing cell lines and NUP214-ABL1-expressing murine bone marrow cells were treated with DPH and Imatinib, as well as Acute Myeloid Leukemia (AML) cells expressing FLT3-ITD mutation when treated with DPH in combination with AC220 which is the FLT3-ITD inhibitor. In summary, ABL1 is a potential tumor-suppressor in BCR-ABL1-induced leukemia and stimulation of its function may play a significant role in the development of novel therapeutic strategies for CML and other Fusion Tyrosine Kinase (FTK)-mediated hematologic malignancies.