Sarah Greenblatt

Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model

Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML). FLT3/ITD mutations have also been observed in myelodysplastic syndrome patients both before and during progression to AML. Previous work has shown that insertion of an FLT3/ITD mutation into the murine Flt3 gene induces a myeloproliferative neoplasm, but not progression to acute leukemia, suggesting that additional cooperating events are required. We therefore combined the FLT3/ITD mutation with a model of myelodysplastic syndrome involving transgenic expression of the Nup98-HoxD13 (NHD13) fusion gene. Mice expressing both the FLT3/ITD and NHD13 transgene developed AML with 100% penetrance and short latency. These leukemias were driven by mutant FLT3 expression and were susceptible to treatment with FLT3 tyrosine kinase inhibitors. We also observed a spontaneous loss of the wild-type Flt3 allele in these AMLs, further modeling the loss of the heterozygosity phenomenon that is seen in human AML with FLT3-activating mutations. Because resistance to FLT3 inhibitors remains an important clinical issue, this model may help identify new molecular targets in collaborative signaling pathways.

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drugs apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.

NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease

Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.

TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo

More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML.

Covalent modifications of RUNX proteins: Structure affects function

The RUNX family of transcription factors plays important roles in tissue-specific gene expression. Many of their functions depend on specific post-translational modifications (PTMs), and in this review, we describe how PTMs govern RUNX DNA binding, transcriptional activity, protein stability, cellular localization, and protein-protein interactions. We also report how these processes can be disrupted in disease settings. Finally, we describe how alterations of RUNX1, or the enzymes that catalyze its post-translational modifications, contribute to hematopoietic malignancies.

Abstract 1807: Characterization of a novel FLT3 inhibitor in AML cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by increased myeloproliferation and a block in differentiation of progenitor cells, leading to infiltration of immature blasts in the bone marrow and peripheral blood. FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic progenitor cells. Approximately 30% of AML cases harbor a mutation in the FLT3 gene leading to constitutive activation of FLT3. The most commonly observed mutation occurs in the juxtamembrane domain as an internal tandem duplication (FLT3-ITD) of variable length sequence repeats. In addition, activating mutations in the kinase domain are observed in 7-10% of patients. The presence of FLT3-ITD mutations confers a poor prognosis, and thus many studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. A number of clinical trials are now underway studying tyrosine kinase inhibitors (TKI) that target FLT3. There have been limitations in the responses observed in patients on these trials related to insufficient achievement of FLT3 inhibition and the development of drug resistance through a variety of mechanisms. This includes resistance conferring point mutations that appear in patients following drug treatment, as well as selection for cells with activation of parallel signaling pathways. Therefore, the search for novel TKIs that overcome some of these mechanisms and the discovery of additional targets for the treatment of AML are necessary to improve the cure rate of this disease. TTT-3002 is a multi-targeted kinase inhibitor that has activity against FLT3-ITD. Here we report the results of the characterization of this compound in a panel of AML cell lines. TTT-3002 has potent activity against FLT3 phosphorylation in FLT3-ITD cell lines at subnanomolar concentrations and significantly decreases cell viability through the induction of apoptosis. Interestingly, TTT-3002 is highly active against cell lines with known activating or resistance mutations in the FLT3 gene, indicating the potential for broad clinical application. Furthermore, through analysis of TTT-3002 potency in cell lines cultured in human plasma preparations from healthy donors, we observed a 10-20 fold shift in IC50 values for FLT3 phosphorylation compared to cell culture media. These results suggest that TTT-3002 has reasonable protein binding, and may require lower doses than many other TKIs to achieve an effective concentration of free drug in plasma. Finally, we have found that 6 mg/kg bid in a solid dosing form is well tolerated in BALB/c mice, and it is sufficient to eliminate luciferase expressing Ba/F3 cells containing a FLT3-ITD mutation after 4 days of treatment. Continual dosing with TTT-3002 significantly prolongs tumor free survival over vehicle control treated mice (p<0.0001, n=10). These findings demonstrate the potential of TTT-3002 as a promising targeted therapeutic in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1807. doi:1538-7445.AM2012-1807