Raghuveer Singh Mali

Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis.

We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04) and sole TET2 mutations (P<0.001). In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

Rho Kinase Regulates the Survival and Transformation of Cells Bearing Oncogenic Forms of KIT, FLT3, and BCR-ABL

We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

Therapeutic Potential of Targeting the Oncogenic SHP2 Phosphatase

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase associated with various kinds of leukemia and solid tumors. Thus, there is substantial interest in developing SHP2 inhibitors as potential anticancer and antileukemia agents. Using a structure-guided and fragment-based library approach, we identified a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than 5-fold selectivity against 20 mammalian PTPs. Structural and modeling studies reveal that the hydroxyindole carboxylic acid anchors the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the β5–β6 loop contribute to 11a-1’s binding potency and selectivity. Evidence suggests that 11a-1 specifically attenuates the SHP2-dependent signaling inside the cell. Moreover, 11a-1 blocks growth factor mediated Erk1/2 and Akt activation and exhibits excellent antiproliferative activity in lung cancer and breast cancer as well as leukemia cell lines.

ROCK1 functions as a critical regulator of stress erythropoiesis and survival by regulating p53

Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.

The PI3K pathway drives the maturation of mast cells via microphthalmia transcription factor

Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1(+)Mac1(+) myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3- or BCR homology domain-deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP(-/-) and p85α(-/-) mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

SH2 Domain-Containing Phosphatase 2 Is a Critical Regulator of Connective Tissue Mast Cell Survival and Homeostasis in Mice

ABSTRACT Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.

Balanced Interactions between Lyn, the p85α Regulatory Subunit of Class IA Phosphatidylinositol-3-Kinase, and SHIP Are Essential for Mast Cell Growth and Maturation

ABSTRACT The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class IA PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyns unique domain. In the absence of Lyns unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.