Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation.

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

The Src family kinase LCK cooperates with oncogenic FLT3/ITD in cellular transformation.

The non-receptor tyrosine kinase LCK belongs to the SRC family of kinases. SRC family kinases are proto-oncogenes that have long been known to play key roles in cell proliferation, motility, morphology and survival. Here we show that LCK regulates the function of the type III receptor tyrosine kinase FLT3 in murine pro-B cells. We observed that expression of LCK significantly enhances the colony forming capacity of the constitutively active FLT3 mutant FLT3-ITD (internal tandem duplication). Furthermore, cells expressing LCK developed tumor earlier compared to cells transfected with empty control vector. Staining of the tissues from mouse xenografts showed higher Ki67 staining in cells expressing LCK suggesting that expression of LCK enhances the FLT3-ITD-mediated proliferative capacity. LCK expression did not affect either FLT3-WT or FLT3-ITD -induced AKT, ERK1/2 or p38 phosphorylation. However, LCK expression significantly enhanced FLT3-ITD-mediated STAT5 phosphorylation. Taken together, our data suggest that LCK cooperates with oncogenic FLT3-ITD in cellular transformation.

Efficacy of the CDK inhibitor dinaciclib in vitro and in vivo in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease of the blood affecting children, adolescents and adults. Although current treatment protocols for T-ALL have improved overall survival, a portion of T-ALL patients still experiences treatment failure. Thus, the development of novel therapies is needed. In this study, we used several patient-derived T-ALL cell lines to screen for an effective drug for T-ALL. Using a panel of 378 inhibitors against different kinases, we identified the CDK inhibitor dinaciclib as a potential drug for T-ALL. Dinaciclib treatment significantly reduced cell viability and completely blocked colony formation. Furthermore, cells treated with dinaciclib showed decreased expression of several pro-survival proteins including survivin, cyclin T1 and c-MYC. Dinaciclib treatment also increased accumulation of cells in G2/M phase and significantly induced apoptosis. Finally, dinaciclib extended survival of mice in a T-ALL cell xenograft model. Collectively, these data suggest that the CDK inhibitor dinaciclib is an active drug for T-ALL in the preclinical settings.

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

The dual specificity PI3K/mTOR inhibitor PKI-587 displays efficacy against T-cell acute lymphoblastic leukemia (T-ALL).

Although significant improvements have been made in the treatment of acute lymphoblastic leukemia (ALL), there is a substantial subset of high-risk T-cell ALL (T-ALL) patients with relatively poor prognosis. Like in other leukemia types, alterations of the PI3K/mTOR pathway are predominant in ALL which is also responsible for treatment failure and relapse. In this study, we show that relapsed T-ALL patients display an enrichment of the PI3K/mTOR pathway. Using a panel of inhibitors targeting multiple components of the PI3K/mTOR pathway, we observed that the dual-specific PI3K/mTOR inhibitor PKI-587 was the most selective inhibitor for T-ALL cells dependent on the PI3K/mTOR pathway. Furthermore, we observed that PKI-587 blocked proliferation and colony formation of T-ALL cell lines. Additionally, PKI-587 selectively abrogated PI3K/mTOR signaling without affecting MAPK signaling both in in vitro and in vivo. Inhibition of the PI3K/mTOR pathway using PKI-587 delayed tumor progression, reduced tumor load and enhanced the survival rate in immune-deficient mouse xenograft models without inducing weight loss in the inhibitor treated mice. This preclinical study shows beneficial effects of PKI-587 on T-ALL that warrants further investigation in the clinical setting.

ABL2 suppresses FLT3-ITD-induced cell proliferation through negative regulation of AKT signaling.

The type III receptor tyrosine kinase FLT3 is one of the most commonly mutated oncogenes in acute myeloid leukemia (AML). Inhibition of mutated FLT3 in combination with chemotherapy has displayed promising results in clinical trials. However, one of the major obstacles in targeting FLT3 is the development of resistant disease due to secondary mutations in FLT3 that lead to relapse. FLT3 and its oncogenic mutants signal through associating proteins that activate downstream signaling. Thus, targeting proteins that interact with FLT3 and their downstream signaling cascades can be an alternative approach to treat FLT3-dependent AML. We used an SH2 domain array screen to identify novel FLT3 interacting proteins and identified ABL2 as a potent interacting partner of FLT3. To understand the role of ABL2 in FLT3-mediated biological and cellular events, we used the murine pro-B cell line Ba/F3 as a model system. Overexpression of ABL2 in Ba/F3 cells expressing an oncogenic mutant of FLT3 (FLT3-ITD) resulted in partial inhibition of FLT3-ITD-dependent cell proliferation and colony formation. ABL2 expression did not alter the kinase activity of FLT3, its ubiquitination or its stability. However, it partially blocked FLT3-induced AKT phosphorylation without affecting ERK1/2 and p38 activation. Taken together our data suggest that ABL2 acts as negative regulator of signaling downstream of FLT3.