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FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)-cell development. CD34(+) hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34(+) HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells.

Entry into the cell cycle is mediated by cyclin-dependent kinase 4/6 (CDK4/6) activation, followed by CDK2 activation. We found that pharmacologic inhibition of the Flt3 internal tandem duplication (ITD), a mutated receptor tyrosine kinase commonly found in patients with acute myelogenous leukemia (AML), led to the down-regulation of cyclin D2 and D3 followed by retinoblastoma protein (pRb) dephosphorylation and G(1) cell-cycle arrest. This implicated the D-cyclin-CDK4/6 complex as a downstream effector of Flt3 ITD signaling. Indeed, single-agent PD0332991, a selective CDK4/6 inhibitor, caused sustained cell-cycle arrest in Flt3 ITD AML cell lines and prolonged survival in an in vivo model of Flt3 ITD AML. PD0332991 caused an initial cell-cycle arrest in well-established Flt3 wild-type (wt) AML cell lines, but this was overcome by down-regulation of p27(Kip) and reactivation of CDK2. This acquired resistance was not observed in a Flt3 ITD and a Flt3 wt sample from a patient with primary AML. In summary, the mechanism of cell-cycle arrest after treatment of Flt3 ITD AML with a Flt3 inhibitor involves down-regulation of cyclin D2 and D3. As such, CDK4/6 can be a therapeutic target in Flt3 ITD AML but also in primary Flt3 wt AML. Finally, acquired resistance to CDK4/6 inhibition can arise through activation CDK2.

The CBL ubiquitin ligase targets a variety of activated tyrosine kinases (TKs) for degradation. Many TKs are mutationally or autocrine activated and/or often overexpressed at the mRNA and protein levels in acute leukemias. We hypothesized that CBL is mutated in patients with acute myeloid leukemia (AML). Four of 12 patients and the MOLM-13 cell line harbored c-CBL mutations, either RNA splicing mutations, missense mutations, or a nucleotide insertion. Additionally, 1 of the 12 patients harbored a missense mutation in the related CBL-b gene. Each c-CBL mutation involves the structurally important alpha-helix within the linker region, while the mutation in CBL-b was located in the Ub-E2 protein-binding RING finger. Short-interfering RNA knockdown of mutant c-CBL present in MOLM-13 cells was growth inhibitory. In summary, novel mutations in c-CBL and CBL-b have been identified in human AML and may represent potential targets for novel therapeutics.