FLT3-ITD AML Research Articles

Different FLT3 Inhibitors Display Limited, Non-Overlapping Profiles of Specific FLT3-ITD Resistance Mutations in a Cell-Based Screen.

Mutations causing resistance to therapeutic kinase inhibition can be identified in target kinases in various malignant diseases, such as Bcr-Abl in CML, FLT3-ITD in AML, cKit in GIST, EGFR in NSCLC, and FIP1L1-PDGFRalpha in HES/CEL. Thus, mutations in target kinases constitute a general mechanism of resistance to therapeutic kinase inhibiton. It has been shown that sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. We therefore intended to determine, whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-ITD expressing cell lines resistant to the FLT3 inhibitors su5614 and PKC412. The frequency of resistant clones per million cells in the presence of inhibitor at 10 times the IC50 was 0.17 for su5614 (1,5?M) and 0 with PKC412 (100nM). When FLT3-ITD transformed cells were mutagenized with ENU (ethylnitrosourea) prior to addition of inhibitors, the frequency of resistant clones increased to 2.33 with su5614, and to 0.34 with PKC412. When we analyzed FLT3-ITD TK1 and TK2 for mutations that may confer inhibitor resistance, we found mutations in 127 out of 179 cell clones (71%) with su5614. Nine different exchanges affecting six positions in TK2 were identified, within or shortly behind the FLT3-ITD A-loop, with exchanges of D835 predominating. We did not detect exchanges affecting TK1. With PKC412, four out of 26 resistant clones (15%) contained mutations. In contrast to su5614, no mutations of TK2 were identified. However, three different amino acid exchanges emerging with PKC412 exclusively affected N676, which is located in TK1 of the FLT3-ITD split kinase domain. This is in line with the observation of a FLT3-ITD N676K exchange identified in a single AML patient with clinical resistance to PKC412 (Heidel et al, 2006). When expressed in cell lines, the identified TK2 A-loop mutations shifted dose-response curves to higher concentrations of su5614, but did not affect response to PKC412, su11248 or sorafenib. However, Y842D caused a strong resistance towards sorafenib, but not to su11248 or PKC412. Recent results also demonstrate a differential response of TK1 N676 exchanges towards different FLT3 inhibitors. We are currently analyzing su11248- and sorafenib- resistant FLT3-ITD clones for specific resistance mutations. Our results demonstrate that cell-based resistance screening is a useful and valid tool for prediction of resistance mutations to kinase inhibitors. In contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib and dasatinib, which display highly overlapping resistance profiles, FLT3 kinase inhibitors may generate distinct, non-overlapping profiles. This opens the possibility of using combined FLT3 inhibitor therapy in AML to prevent resistance due to FLT3-ITD kinase domain mutations.

An Orally Bioavailable Inhibitor of FLT3 and Syk Kinases Prevents Tumor Growth in Subcutaneously Implanted Human Tumor Xenografts and Promotes Cell Death of FLT3 Mutant AML Cells.

Background. Phase 1 clinical studies have shown that an orally bioavailable syk kinase inhibitor, R788/406, is very well tolerated in human volunteers for up to 7 days (and in primates for up to 28 days) at doses achieving concentrations in excess of 5 micromolar (ED50 for in vivo biomarker of syk-inhibition in humans is 1 micromolar). In biochemical kinase assays, R788/406 inhibits syk and FLT3 at less than 10 nM, and in cell-based assays at less than 100 nM. The demonstration of biological activity and excellent tolerability in humans, and the equipotent inhibition of FLT3 and syk kinases in biochemical assays led us to explore the potential for use of R788/406 in a xenograft of a human AML FLT-3ITD mutant cell line.Objective: To evaluate the in vitro and in vivo activity of R788/406 in a human AML FLT3-ITD mutant cell line, MV411, a model system for examination of FLT3 mutant AML.Methods: MV411 cells were treated with R788/406 and evaluated for cell viability and markers of apoptosis (Annexin-V/PI and caspase) by FACS. Cell cycle analysis was performed on cells stained with PI. 5 X 106 MV411 cells harvested in logarithmic phase growth were injected with Matrigel SC in NOD/SCID mice. Treatment with R788/406 began when tumors reached a predetermined size (mean volume of 100 mm3) and continued for 26 days. At sacrifice, tumor xenografts were lysed, immunoprecipitated with anti-FLT-3, and probed with anti-phosphotyrosine 4G10 or anti-FLT-3.Results: R788/406 potently and selectively induced dose-dependent cytotoxicity of MV-411 AML cells in vitro with an ED50 of 20nM. Pretreatment of cells with R788/406 promoted dephosphorylation of constitutively active pFLT3, as well as a reduction of pStat5 and pErk1/2 in the FLT3 signaling cascade. Moreover, R788/406 induced cell cycle arrest in the G1 phase and subsequent apoptosis in MV411 cells in a dose-dependent manner. Twice daily administration of R788/406 to NOD/SCID mice bearing SC MV411 tumors reduced tumor growth significantly in a dose dependent manner. When compared to vehicle controls, daily doses of 40 and 80mg/kg R788/406 resulted in 45% and 82% inhibition of mean tumor volumes, respectively. At study termination mean tumor volumes were 686.90 ± 115.56 and 224.45 ± 49.80 for 40 and 80 mg/kg R788/406-treated animals compared to 1255.48 ± 182.94 for vehicle controls with a final %T/C of -0.4 (range of %T/C throughout study was −7.9 to −0.4). During the study, no significant body weight loss was observed in any of the animals in this study. Ex vivo analyses of subcutaneous tumors from MV411 tumor-bearing mice showed that R788/406 completely inhibited constitutive FLT3 activation and downstream signaling events.Conclusions: R788/406 is well tolerated in humans (and primates) at concentrations well in excess of those that inhibit syk in vivo. Given the equipotent inhibition of syk and FLT3, the in vivo activity against human syk, and the xenograft data reported here, R788/406 may be a promising agent for FLT-3 AML.

Dissection of Submicroscopical Aberrations and Clonal Evolution from Birth To Diagnosis and Relapse in a Childhood AML FLT3-ITD Positive Patient by RQ-PCR and SNP Array Analyses.

We have performed a combined Real Time Quantitative-PCR and single nucleotide polymorphisms array analyses for dissecting the clonal evolution in a childhood AML patient who experienced two relapses and for whom we had the availability of the cord blood (CB) sample. The patient was diagnosed at 6 years of age with an AML-M1 and showed normal karyotype and a FLT3-ITD mutation as a sole abnormality. She underwent autologous-BMT; however, 3.5 months later the patient relapsed, and 4 months after an allogeneic-BMT she suffered from a second relapse and died for disease progression. Highly sensitive (10−4) monitoring of FLT3-ITD was performed by patient-specific RQ-PCR, and showed a progressive decrease of minimal residual disease (MRD) during induction therapy. MRD was below the detection limit before auto-BMT, but the same FLT3-ITD clone re-emerged three months after auto-BMT, and preceded the clinical relapse. Thus, the same FLT3-ITD mutation was detected at the time of relapse, suggesting that the leukemic clone responsible for the first diagnosis was still present and could be potentially used as a marker for backtracking the leukemia into the CB. When tested by highly sensitive RQ-PCR, the DNA from CB resulted negative for the FLT3-ITD mutation. Although the relatively limited sensitivity of the technique might impair the interpretation, the FLT3-ITD negative result in CB is consistent with the hypothesis that FLT3-ITD mutations are secondary events, not sufficient by themselves to induce leukemia transformation in hematopoietic stem cells without a necessary primary event. With the aim to find additional submicroscopical genetic changes associated to the highly aggressive nature of the patient disease, we performed a genome wide SNP array analysis on the patient DNA through the clinical evolution of the disease, from birth to relapse. This new SNP array strategy is emerging as a powerful method to detect loss of heterozygosity (LOH) and/or copy number changes in a DNA sample with high resolution. The Affymetrix GeneChip® Mapping 10K platform has been used, which allows the scanning of more than 10.000 SNPs. SNP array analysis on DNA from the first relapse showed the deletion of the long arm of chromosome 9, a recurring chromosomal aberration in AML, and LOH on the whole chromosome 13 not associated with copy number changes. This latter has been confirmed by FISH, and it is consistent with uniparental isodisomy (UPD) as a responsible mechanism for the somatically acquired homozygosity of FLT3-ITD at 13q14. This mechanism is emerging as a frequent way of disease progression and represents a subsequent event to FLT3-ITD heterozygous mutation. The deletion of the wild type FLT3 allele has been confirmed by PCR. 10K SNP array analysis failed to reveal LOH or copy number changes in the diagnostic and in CB samples. These findings are compatible with a somatic post-natal origin of the FLT3-ITD positive AML subtype. Additional abnormalities can be responsible for the disease progression, via different mechanisms, including UPD. Other methods must be applied to find the primary event(s) giving rise to leukemia in association with FLT3-ITD mutation.

FLT3 Internal Tandem Duplications in Adults with De Novo Acute Myeloid Leukemia.

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. Recently, internal tandem duplication (ITD) mutations of the FLT3 gene have been described in patients with AML and associated with a poor prognosis. The aim of this study was to analyze the prevalence of FLT3-ITD in a series of 90 adults with de novo AML and correlate the presence of this mutation with biological characteristics and clinical response. We analyzed diagnostic peripheral blood or bone marrow specimens from 43 women and 47 men, with a median age of 38 years (16–83). Polymerase chain reaction was performed on genomic DNA using previously published primers for exons 11 and 12. An FLT3-ITD was found in 22/89 patients (25%). It was present in 37% (9/24) of the patients with acute promyelocytic leukemia (APL) and in only 20% (13/65) of the patients with non-M3-AML (p=0.07). The FLT3-ITD was not detected in patients with M6 (n=1) and M7-AML (n=3), nor in patients with the AML1-ETO (n=2) or with the CBFb-MYH11 (n=4) fusion genes. The median WBC counts were higher in FLT3-ITD patients than in those without the mutation (37 X 109/L vs. 27 X 109/L, p=0.43). In APL, FLT3-ITD was found in 5 out of 6 patients with the short PML-RARa isoform, but in only 4 out of 18 patients with the non-short isoform (p=0.01). Univariate analysis showed an association between the presence of FLT3-ITD and both a lower complete remission (CR) rate (41% vs. 64%; p=0.05) and a shorter overall survival (14% vs. 34%; p=0.03). However, FLT3-ITD was not associated with the CR rate (p=0.18) or the OS (p=0.07) in the multivariate analysis. The clinical significance of FLT3-ITD in adult AML remains uncertain, and further investigation is clearly warranted.