Role of Rho kinases in abnormal and normal hematopoiesis

PURPOSE OF REVIEW: Rho kinases (ROCKs) are involved in regulating a variety of physiologic functions including cytoskeletal reorganization, migration, adhesion, survival and proliferation. They do so via activating several different downstream substrates such as myosin light chain phosphatase, LIM kinase and ezrin/radixin/moesin proteins. To date, most of the conclusions with regard to the function of ROCKs have involved the use of cell line models, pharmacologic inhibitors and dominant negative approaches. Importantly, the role of ROCK in hematopoiesis or leukemogenesis in the context of whole organism remains poorly understood. RECENT FINDINGS: Recent studies utilizing mice deficient in the expression of ROCK1 have begun to shed some light into the physiologic role(s) of ROCK in both normal and abnormal hematopoiesis. Findings, thus far, suggest that ROCK plays an essential role in regulating growth and survival in different hematopoietic lineages via distinct mechanisms, in part, by utilizing distinct downstream substrates including maintaining the activation of tumor-suppressor genes. SUMMARY: In blood cells, emerging data suggest that ROCK plays an essential role in negatively regulating inflammatory and erythropoietic stress and positively regulates the growth and survival of leukemic cells

Targeting phosphatidylinositol-3-kinase pathway for the treatment of Philadelphia-negative myeloproliferative neoplasms

Myeloproliferative neoplasms (MPN) are a diverse group of chronic hematological disorders that involve unregulated clonal proliferation of white blood cells. Sevearl of them are associated with mutations in receptor tyrosine kinases or cytokine receptor associated tyrosine kinases rendering them independent of cytokine-mediated regulation. Classically they have been broadly divided into BCR-ABL1 fusion + ve (Ph + ve) or -ve (Ph-ve) MPNs. Identification of BCR-ABL1 tyrosine kinase as a driver of chronic myeloid leukemia (CML) and successful application of small molecule inhibitors of the tyrosine kinases in the clinic have triggered the search for kinase dependent pathways in other Ph-ve MPNs. In the past few years, identification of mutations in JAK2 associated with a majority of MPNs raised the hopes for similar success with specific targeting of JAK2. However, targeting JAK2 kinase activity has met with limited success. Subsequently, mutations in genes other than JAK2 have been identified. These mutations specifically associate with certain MPNs and can drive cytokine independent growth. Therefore, targeting alternate molecules and pathways may be more successful in management of MPNs. Among other pathways, phosphatidylinositol -3 kinase (PI3K) has emerged as a promising target as different cell surface receptor induced signaling pathways converge on the PI3K signaling axis to regulate cell metabolism, growth, proliferation, and survival. Herein, we will review the clinically relevant inhibitors of the PI3K pathway that have been evaluated or hold promise for the treatment of Ph-ve MPNs

Role of mTORC1-S6K1 signaling pathway in regulation of hematopoietic stem cell and acute myeloid leukemia

Dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1)-p70 ribosomal protein kinase 1 (S6K1) signaling pathway occurs frequently in acute myeloid leukemia (AML) patients. This pathway also plays a critical role in maintaining normal cellular processes. Given the importance of leukemia stem cells (LSCs) in the development of minimal residual disease, it is critical to use therapeutic interventions that target the LSC population to prevent disease relapse. The mTORC1-S6K1 pathway has been identified as an important regulator of hematopoietic stem cell (HSC) and LSC functions. Both HSC and LSC functions require regulation of key cellular processes including proliferation, metabolism, and autophagy, which are regulated by mTORC1 pathway. Despite the mTORC1-S6K1 pathway being a critical regulator of AML initiation and progression, inhibitors of this pathway alone have yielded mixed results in clinical studies. Recent studies have identified strategies to develop new mTORC1-S6K1 inhibitors such as RapaLink-1, which could circumvent the drug resistance observed in AML cells and in LSCs. Here, we review recent advances made in identifying the role of different components of this pathway in the regulation of HSCs and LSCs and discuss possible therapeutic approaches

Mastocytosis: a mutated KIT receptor induced myeloproliferative disorder

Although more than 90% systemic mastocytosis (SM) patients express gain of function mutations in the KIT receptor, recent next generation sequencing has revealed the presence of several additional genetic and epigenetic mutations in a subset of these patients, which confer poor prognosis and inferior overall survival. A clear understanding of how genetic and epigenetic mutations cooperate in regulating the tremendous heterogeneity observed in these patients will be essential for designing effective treatment strategies for this complex disease. In this review, we describe the clinical heterogeneity observed in patients with mastocytosis, the nature of relatively novel mutations identified in these patients, therapeutic strategies to target molecules downstream from activating KIT receptor and finally we speculate on potential novel strategies to interfere with the function of not only the oncogenic KIT receptor but also epigenetic mutations seen in these patients

Role of SHP2 in hematopoiesis and leukemogenesis

Purpose of review SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11 plays an important role in regulating signaling from cell surface receptor tyrosine kinases during normal development as well as oncogenesis. Herein we review recently discovered roles of SHP2 in normal and aberrant hematopoiesis along with novel strategies to target it. Recent findings Cell autonomous role of SHP2 in normal hematopoiesis and leukemogenesis has long been recognized. The review will discuss the newly discovered role of SHP2 in lineage specific differentiation. Recently, a noncell autonomous role of oncogenic SHP2 has been reported in which activated SHP2 was shown to alter the bone marrow microenvironment resulting in transformation of donor derived normal hematopoietic cells and development of myeloid malignancy. From being considered as an ‘undruggable’ target, recent development of allosteric inhibitor has made it possible to specifically target SHP2 in receptor tyrosine kinase driven malignancies. Summary SHP2 has emerged as an attractive target for therapeutic targeting in hematological malignancies for its cell autonomous and microenvironmental effects. However a better understanding of the role of SHP2 in different hematopoietic lineages and its crosstalk with signaling pathways activated by other genetic lesions is required before the promise is realized in the clinic

Rap1 is a Potential Therapeutic Target for Non-myeloablative Conditioning

poster abstractvarious side effects including gastrointestinal mucositis. Identification of therapeutic targets and determining their role in HSC development and function is important to determine a regimen for nonmyeloablative conditioning. Previous studies have shown that GTPases play a critical role in self-renewal, engraftment and retention of HSCs. Rap1, a GTPase, is necessary for migration, adhesion as well as function of mature hematopoietic cells. To study the role of Rap1 in hematopoietic stem and progenitor cells (HSC/Ps), we have generated a mouse model in which the Rap1a and Rap1b isoforms of Rap1 are conditionally deleted in HSC/Ps (Rap1a/b -/-). Deficiency of Rap1a/b results in increased peripheral blood count as well as increase in HSCs in bone marrow along with a decrease in bone marrow cellularity. Rap1a/b deficient bone marrow HSC/Ps also have reduced adhesion capability in vitro. The self-renewal property of HSCs, in conjunction with their ability of multi-lineage reconstitution is important to repopulate the hematopoietic system of irradiated recipients of bone marrow transplant. Rap1a/b -/- HSCs show a defect in engraftment as well as multi-lineage reconstitution when they are transplanted into lethally irradiated hosts. Rap1 deficient HSCs show decreased homing into bone marrow of lethally irradiated recipients. To determine whether Rap1 can be used as a potential target for nonmyeloablative conditioning, we performed bone marrow transplant into WT and Rap1a/b -/- mice without prior irradiation. Deficiency of Rap1a/b in HSCs resulted in availability of bone marrow niche for exogenously transplanted HSCs to engraft along with subsequent multi-lineage reconstitution. Overall, our study reveals that Rap1a/b are important for homing and retention of hematopoietic cells in bone marrow and deletion of Rap1a/b in HSCs result in engraftment of exogenous HSCs within the bone marrow of non-irradiated recipients

COOPERATION OF AML1-ETO AND ONCOGENIC KIT IN ACUTE MYELOGENOUS LEUKEMIA

poster abstractA significant portion of AML patients have the cytogenetic abnormality t(8;21) which generates the fusion protein AML1-ETO, leading to a disruption of the core binding factor complex that regulates transcription of hematological genes. Patients harboring the translocation alone usually have a good prognosis; however, a substantial portion of patients bearing an additional oncogenic receptor tyrosine kinase, KIT, mutation have significantly worse prognosis. A mutation of aspartic acid to valine (KITD814V) in the activation loop results in altered substrate recognition and utilization, constitutive tyrosine autophosphorylation, and promiscuous signaling. Little is known concerning possible mechanisms of cooperation between AML1-ETO and KITD814V. Using an IL3 dependent murine myeloid cell line, we show that growth of AML1-ETO bearing cells remain ligand dependent, while cells that express both AML1-ETO and KITD814V demonstrate ligand independent proliferation. Furthermore, functional assays show that expression of AML1-ETO and KITD814V leads to an increase in cell cycling and decrease in apoptosis that may contribute to the observed ligand independent proliferation. Using a syngenic murine transplantation model we demonstrate that mice transplanted with AML1-ETO and KITD814V bearing cells succumb to a fatal myeloproliferative disease (MPD)-like phenotype, while AML1-ETO expressing mice remain disease free. This suggests that AML1-ETO alone is not sufficient to induce ligand independent growth, nor MPD, but may cooperate with KITD814V to enhance proliferation. Continuing research aims to investigate mechanisms of cooperation between KITD814V and AML1-ETO that contribute to ligand independent growth in vitro, transformation in vivo, and poor overall prognosis in AML patients bearing the two mutations

Imipramine blue sensitively and selectively targets FLT3-ITD positive acute myeloid leukemia cells.

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200-300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75-150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML