Hardik Modi

Enhanced BCR-ABL kinase inhibition does not result in increased inhibition of downstream signaling pathways or increased growth suppression in CML progenitors

The therapeutic success of imatinib in chronic myeloid leukemia (CML) is hampered by persistence of malignant stem cells. We investigated whether nilotinib, a more potent BCR-ABL kinase inhibitor could target CML primitive progenitors more effectively than imatinib. CML and normal progenitor cells were cultured with nilotinib or imatinib in growth factor supplemented medium. Nilotinib inhibited BCR-ABL kinase activity at lower concentrations than imatinib. Nilotinib inhibited mitogen-activated protein kinase (MAPK), AKT and STAT5 phosphorylation in CML CD34+ cells in the absence of growth factors (GFs), but did not suppress AKT and STAT5 activity, and resulted in increased MAPK activity, in the presence of GFs. Nilotinib and imatinib resulted in similar suppression of CML primitive and committed progenitors in long-term culture-initiating cell and colony-forming cell assays. Inhibition of progenitor growth was related to marked reduction in proliferation, but only a modest increase in apoptosis. Nilotinib did not show increased efficacy in reducing nondividing CML progenitors compared with imatinib. These results indicate that more potent tyrosine kinase inhibitors by themselves will not be more effective in eliminating CML progenitors than imatinib and that additional mechanism required for maintenance of malignant stem cells need to be identified to improve targeting of leukemia stem cells.

Activation of stress response gene SIRT1 by BCR-ABL promotes leukemogenesis

The tyrosine kinase inhibitor imatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but primary and acquired resistance of CML cells to the drug offset its efficacy. Molecular mechanisms for resistance of CML to tyrosine kinase inhibitors are not fully understood. In the present study, we show that BCR-ABL activates the expression of the mammalian stress response gene SIRT1 in hematopoietic progenitor cells and that this involves STAT5 signaling. SIRT1 activation promotes CML cell survival and proliferation associated with deacetylation of multiple SIRT1 substrates, including FOXO1, p53, and Ku70. Imatinib-mediated inhibition of BCR-ABL kinase activity partially reduces SIRT1 expression and SIRT1 inhibition further sensitizes CML cells to imatinib-induced apoptosis. Knockout of SIRT1 suppresses BCR-ABL transformation of mouse BM cells and the development of a CML-like myeloproliferative disease, and treatment of mice with the SIRT1 inhibitor tenovin-6 deters disease progression. The combination of SIRT1 gene knockout and imatinib treatment further extends the survival of CML mice. Our results suggest that SIRT1 is a novel survival pathway activated by BCR-ABL expression in hematopoietic progenitor cells, which promotes oncogenic transformation and leukemogenesis. Our findings suggest further exploration of SIRT1 as a therapeutic target for CML treatment to overcome resistance.

Janus kinase 2 regulates Bcr-Abl signaling in chronic myeloid leukemia.

Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

A critical role for SHP2 in STAT5 activation and growth factor-mediated proliferation, survival, and differentiation of human CD34 cells

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.

Chronic myelogenous leukemia stem and progenitor cells demonstrate chromosomal instability related to repeated breakage-fusion-bridge cycles mediated by increased nonhomologous end joining

Chromosomal aberrations are an important consequence of genotoxic exposure and contribute to pathogenesis and progression of several malignancies. We investigated the susceptibility to chromosomal aberrations in chronic myelogenous leukemia (CML) progenitors after exposure to ionizing radiation. In normal progenitors, ionizing radiation induced both stable and unstable chromosomal lesions, but only stable aberrations persisted after multiple divisions. In contrast, radiation of chronic phase CML progenitors resulted in enhanced generation of unstable lesions that persisted after multiple divisions. CML progenitors demonstrated active cell cycle checkpoints and increased nonhomologous end joining DNA repair, suggesting that persistence of unstable aberrations was the result of continued generation of these lesions. CML progenitors demonstrated enhanced susceptibility to repeated cycles of chromosome damage, repair, and damage through a breakage-fusion-bridge mechanism. Perpetuation of breakage-fusion-bridge cycles in CML progenitors was mediated by classic nonhomologous end joining repair. These studies reveal a previously unrecognized mechanism of chromosomal instability in leukemia progenitors because of continued generation of unstable chromosomal lesions through repeated cycles of breakage and repair of such lesions.

Inhibition of Grb2 expression demonstrates an important role in BCR-ABL-mediated MAPK activation and transformation of primary human hematopoietic cells

Chronic myeloid leukemia (CML) results from the expression of the BCR/ABL oncogene in a primitive hematopoietic cell. However, BCR/ABL-activated signaling mechanisms are dependent on the cellular context in which it is expressed, and mechanisms underlying primitive human hematopoietic cell transformation by BCR–ABL are not well understood. Our previous studies have shown that BCR/ABL-Y177 has an essential role in Ras activation and human hematopoietic progenitor transformation in CML. The adapter protein growth factor receptor-binding protein-2 (Grb2) can bind phosphorylated BCR/ABL-Y177, induce Grb2-SoS complex formation and activate Ras signaling. We investigated the role of Grb2 in CML progenitor transformation by cotransducing human CD34+ cells with lentivirus vectors expressing short hairpin RNA to Grb2 and retrovirus vectors expressing BCR/ABL. We show that Grb2 knockdown significantly inhibits proliferation and survival of BCR–ABL-expressing CD34+ cells, but not control CD34+ cells. Grb2 knockdown reduced mitogen-activated protein kinase (MAPK) activity in BCR–ABL-expressing hematopoietic cells. We conclude that inhibition of Grb2 expression demonstrates an important role in BCR–ABL-mediated MAPK activation and transformation of primary human hematopoietic cells.These results support further investigation of downstream effectors of Grb2-mediated signals and targeting of Grb2 interactions in the treatment of CML.

Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells.

OBJECTIVE This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells. MATERIALS AND METHODS shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining. RESULTS Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells. CONCLUSION These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.

Abstract B46: Essential roles of SIRT1 for BCR‐ABL transformation and genetic mutations

Resistance of cancer stem cells and acquired genetic mutations are daunting challenges facing targeted cancer therapy, but the underlying mechanisms are not clear. This is best exampled by the tyrosine kinase inhibitor imatinib that effectively treats chronic myelogenous leukemia (CML) whereas resistance develops due to persistence of CML stem/progenitor cells and acquired mutations of BCR‐ABL. SIRT1 is a mammalian stress response gene homologous to yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylase, and is involved in regulation of a variety of cellular functions including survival, glucose homeostasis and fat metabolism through deacetylating histones and non‐histone target proteins. Here we demonstrate that BCRABL transformation activates SIRT1 in hematopoietic stem/progenitor cells in both clinical CML patients and a CML‐like disease mouse model. Inhibition of SIRT1 by gene knockdown or pharmacological inhibitors suppresses CML cell proliferation and induces apoptosis in vitro. SIRT1 knockdown inhibits growth of CML tumor xenograft in immunodeficient mice. Homozygous knockout of SIRT1 gene inhibits BCR‐ABL transformation of mouse bone marrow cells and the development of a CML‐like myeloproliferative disease. We show that through deacetylation of Ku70, SIRT1 promotes BCR‐ABL mutations upon imatinib treatment and de novo genetic mutations upon DNA damage. Our results suggest an essential role of SIRT1 for BCR‐ABL oncogenic transformation and promoting cancer genome instability, and have implication for targeting SIRT1 to overcome resistance. These findings not only improve our understanding of SIRT1 functions in tumorigenesis, but also have translational implication for targeting SIRT1 for overcoming resistance in cancer treatment. Citation Information: Cancer Res 2009;69(23 Suppl):B46.