Including Internal Tandem Duplication Research Articles

T-Cell Receptor-Engineered T Cells Targeting FLT3-D835 Mutation-Derived Neoantigens in Acute Myeloid Leukemia

The adoptive transfer of T-cell receptor (TCR)-engineered T cells has demonstrated a remarkable anti-leukemic potential in mice models and human research for acute myeloid leukemia (AML). Neoantigens arising from somatic mutations are ideal targets for immunotherapy due to their high immunogenicity and harmless to normal cells. Therefore, neoantigen-specific TCR-based immunotherapy may have great prospects for AML. Activating mutations in FMS-like tyrosine kinase 3 ( FLT3) represent the most frequent genomic alterations of AML, including internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain, mostly at the activation loop residue D835. A number of FLT3 inhibitors have been clinically employed for FLT3-mutated AML. However, a durable response is limited by resistance-conferring secondary kinase domain mutations, notably point mutations at D835. Therefore, there is a critical need for novel and potent treatments to eliminate AML cells harboring FLT3-D835 mutations. Here, we show the construction of TCR-engineered T cells targeting FLT3-D835 mutants, presented in the context of HLA-A *02:01. We first identified neoepitopes derived from D835Y/H/V/F/I with strong affinities to HLA-A *02:01 using NetMHCpan 4.1. These neoepitopes could form the stable complex with HLA-A *02:01 on the cell surface. Furthermore, CD8 + T cells specific for D835H were detected in the peripheral blood of AML patients carrying D835H mutant at different stages of the illness, most of which were memory T cells. Moreover, one neoepitope (Neo-D835H) induced IFN-γ secretion when co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from AML patients with D835H mutant. Neo-D835H-specific T cells were effectively generated through the rapid stimulation of CD8 + T cells isolated from an HLA-A *02:01-positive healthy donor with Neo-D835H-loaded autologous dendritic cells. These T cells were recognized by Neo-D835H-HLA-A2-Tetramer (0.37%) and consequently isolated by flow cytometry to perform TCRα and TCRβ sequencing. Both the TCRA and TCRB clonotypes showed dominant sequences that accounted for more than 50% of the total, suggesting that T cells with these dominant TCRA and TCRB-paired sequences were likely Neo-D835H-specific. We thus next generated TCR-engineered T cells with these TCRA- and TCRB-paired sequences for further functional analysis. These TCR-engineered T cells recognized Neo-D835H and showed cytotoxic activity against Neo-D835H-pulsed T2 cells in an antigen dose-dependent manner, while showed no responses to the T2 cells pulsed with the corresponding wild-type peptide. Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

Secondary Lesions and Sensitivity to Signaling Inhibitors of Pediatric iAMP21 B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study we aimed to determine the frequency of secondary lesions and investigated the cellular sensitivity for candidate targeted drugs. Methods We performed total RNA sequencing on 28 iAMP21 and 28 B-other (negative for sentinel fusion genes) pediatric samples to determine the frequency of secondary lesions in newly diagnosed patients. A panel of 18 patient derived xenografts (PDX) of 8 primary iAMP21 ALL samples was generated, and secondary lesions were validated by PCR, RT-PCR, and whole exome sequencing. To test sensitivity, primary or PDX cells were exposed ex vivo to a concentration range of gilteritinib (FLT3 inhibitor), trametinib (MEK1/2 inhibitor), or ruxolitinib (JAK1/2 inhibitor). Results Secondary lesions in the cytokine receptor gene FLT3 were enriched in iAMP21 compared with B-other ALL including internal tandem duplications (ITD) and other activating lesions (50.0% vs. 10.7%, p=0.003). Lesions in genes encoding cytokine receptors CRLF2 and IL7R had a similar frequency between iAMP21 and B-other cases (25% vs. 17.9%, p=0.75 and 7.1% vs. 0%, p=0.49 respectively). Inactivating lesions in SH2B3, the downstream negative regulator of JAK/STAT and FLT3 signalling, were more frequent in iAMP21 cases vs. B-other (46.4% vs. 7.1%, p=0.002), while the frequency of JAK1 and JAK2 mutations did not differ (3.6 vs. 0% and 10.7 vs. 10.7% p = 1 for both). All SH2B3, CRLF2 and JAK lesions were retained in PDX samples, whereas in contrast FLT3-ITD was retained in only 2 of 5 PDX. Gilteritinib sensitivity did not differ between iAMP21 and B-other cases (median LC50 1.24 µM, range 0.33 to 4.85 µM vs. median LC50 1.21 µM, range 1.20 to 1.3 µM, p=0.95). Grouping iAMP21 cases by FLT3 and SH2B3 status, samples with both FLT3-ITD and SH2B3 lesion had the highest sensitivity to gilteritinib (median LC50 0.39 µM, range 0.35 to 0.43 µM). Samples with only FLT3 or SH2B3 lesion did not show increased sensitivity compared to those without a lesion (p>0.5 for both). Median ruxolitinib sensitivity did not statistically differ between iAMP21 and B-other cases (median LC50 8.38 µM, range 1.00 to >10 µM, vs. median LC50 0.48, range 0.21 to >10 µM; p=1) although extreme resistance to ruxolitinib seemed more frequent in iAMP21 cases (6 out of 12 cases) and was not related to FLT3 or SH2B3 status. CRLF2-rearranged ( CRLF2r) iAMP21 cases were slightly more sensitive to ruxolitinib than those lacking CRLF2r, although this difference did not reach significance (median LC50 4.72 µM, range 1.0-4.85 vs. median LC50 >10 µM, range 2.66 to >10 µM; p=0.08). The highest sensitivity was found in the only case with both a CRLF2r and a JAK1 mutation (LC50 of 1.00 µM). A large variation was present in trametinib sensitivity amongst both iAMP21 and B-other cases (median LC50 0.11 µM, range 0.017 to >5 µM vs. median LC50 0.18, range 0.018 to >5 µM; p=0.72). More than half of iAMP21 cases were sensitive irrespective of RAS-pathway lesion status. Conclusion iAMP21 leukemias are enriched in FLT3 and in SH2B3 lesions which, when co-occurring, affect sensitivity to FLT3 inhibition by gilteritinib but do not affect JAK-inhibition by ruxolitinib. This suggests these lesions act synergistically and might bypass downstream JAK/STAT signalling. This might also explain the observed sensitivity to RAS-pathway inhibition irrespective of secondary lesions. These results suggest that further research into FLT3 and RAS signalling inhibitors might lead to better treatment options for pediatric iAMP21 BCP-ALL.

Mutation spectrum of FLT3 and significance of non-canonical FLT3 mutations in haematological malignancy.

Fms-like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non-canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non-canonical FLT3 mutations depending on the affected protein structure: namely non-canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high-frequency (>1%) FLT3-NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3-deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3-ITD mutant of TKD2 had significantly higher kinase activity than wild-type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild-type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non-canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non-canonical mutations.

Abstract 5456: Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations

Abstract FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5456.

Soft Tissue and Visceral Organ Sarcomas With BCOR Alterations.

Sarcomas with BCOR alteration are a heterogenous group characterized by changes including internal tandem duplications (ITDs) and recurring fusions with CCNB3, ZC3H7B, and other rare partners. With widespread genomic testing, these alterations are now associated with histologies such as Ewing-like sarcoma (BCOR::CCNB3), high-grade endometrial stromal sarcoma (ZC3H7B::BCOR), and clear cell sarcoma of kidney (BCOR-ITD). BCOR altered sarcomas of soft tissues and organs were identified through PubMed using keywords "Sarcoma (AND) BCOR" from 2005 through October 2021. Summary statistics and outcome data were calculated using STATA v12.1. Forty-one publications described 190 patients with BCOR altered soft tissue or organ sarcomas. BCOR-ITD was most common, followed by BCOR::CCNB3, ZC3H7B::BCOR. BCOR-ITD tumors occurred mainly in infants, BCOR::CCNB3 commonly occurred in adolescent young adults, and ZC3H7B::BCOR only in adults. The most common site for BCOR::CCNB3 fused tumors was extremity, BCOR-ITD kidney and ZC3H7B::BCOR uterus. Metastasis was rare in patients with BCOR::CCNB3. While most underwent resection and chemotherapy, few received radiation. Median follow-up of survivors was 24 months. Five year overall survival for patients with BCOR::CCNB3 fusions was 68% (95% confidence interval [CI]: 46%-83%). Patients with BCOR-ITD and ZC3H7B::BCOR had worse prognoses with 5 years overall survival of 35% (95% CI: 15%-56%) and 41% (95% CI: 11%-71%), respectively, demonstrating need for collaborative efforts identifying optimal treatments to improve outcomes.

A Phase 1, Dose-Escalation Study of Gilteritinib Combined with Chemotherapy in Patients Aged 6 Months to

Background: Acute myeloid leukemia (AML) accounts for ~18% of all childhood leukemias (Puumala SE, et al. Pediatr Blood Cancer. 2013;60(5):728-733). Gilteritinib, an oral FMS-like tyrosine kinase 3 (FLT3) inhibitor, has demonstrated efficacy with favorable tolerability in clinical trials of adults with FLT3-mutated relapsed or refractory (R/R) AML, including internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations (Perl AE, et al. N Engl J Med. 2019;381(18):1728-1740), leading to approval for the treatment of adults with R/R AML in the United States and many other countries/regions. Since the clinical progression of FLT3 ITD AML appears to be similar in children and adults, it is expected that the clinical benefit of gilteritinib in pediatric patients with FLT3 ITD R/R AML should be similar to that demonstrated in adults. We describe an ongoing phase 1, multicenter, open-label, single-arm, dose-escalation study (ClinicalTrials.gov identifier: NCT04240002) investigating the combination of gilteritinib with chemotherapy in children, adolescents, and young adults with FLT3 ITD R/R AML, which will inform the dose for the phase 2 dose-expansion portion of the study.Study Design and Methods: This phase 1 dose-escalation study will enroll patients 6 months to <21 years of age with FLT3 ITD and/or TKD-mutated AML (determined using institutional assay). Key inclusion and exclusion criteria include patients who are in first or greater relapse or are refractory to induction therapy with no more than 1 attempt at remission induction and (a) fully recovered from the acute toxic effects of all prior therapy, (b) have a Karnofsky (if ≥16 years) or Lansky (if <16 years) score ≥50, and (c) been ≥90 days relapsed since hematopoietic stem cell transplantation with no active graft-versus-host disease, if relevant. Patients will undergo screening, including complete physical exam, subject-reported symptoms, and areas of disease (Figure). Patients will be enrolled in 1 of the 3 following groups: group 1 (2 to <21 years), group 2 (1 to <2 years), and group 3 (6 months to <1 year). Gilteritinib will be administered once daily starting from day 8 through day 21 of a 28-day cycle at the starting dose of 2 mg/kg/day for Group 1 and 1 mg/kg/day for Groups 2 and 3, with escalation to 2 or 3 mg/kg/day (the latter to be evaluated only if there is lack of toxicity or acceptable dose-limiting toxicity [DLT] and lack of sufficient gilteritinib activity observed at 2 mg/kg/day). Patients in all groups will receive FLAG (fludarabine, 30 mg/m 2/day; cytarabine, 2000 mg/m 2/day; granulocyte-colony stimulating factor, 5 μg/kg/day) on days −1 to 5 and prophylactic single intrathecal cytarabine (age-adjusted dose). Patients who complete 2 cycles of treatment will have the option to participate in long-term treatment (LTT) with gilteritinib for ≤2 years. An end of treatment (EOT) visit will occur 7 days after the last dose of gilteritinib or before initiation of another anticancer therapy. A follow-up visit will be performed 28 days after the EOT visit in patients who are ineligible for LTT. Patients eligible for LTT will undergo a follow-up visit 28 days after the LTT EOT visit. Follow-up for survival will be performed every 3 months for ≤2 years from the 28-day follow-up visit.Study End Points: The primary end point is identification of the maximum tolerated dose and/or recommended phase 2 dose based on the DLT observed in treatment cycle 1 and biologic activity according to plasma inhibitory activity (PIA). The secondary end points are inhibition of phosphorylated FLT3, gilteritinib pharmacokinetics, safety, tolerability, toxicity, event-free survival, overall survival, minimal residual disease assessment, and acceptability and palatability assessment of the formulation. The exploratory end points are correlation of clinical responses to gilteritinib with FLT3 PIA levels, correlation of FLT3 PIA levels and clinical responses to gilteritinib therapy with FLT3 ligand levels before and after exposure to FLAG chemotherapy, and mechanisms of innate and acquired resistance to gilteritinib. All data will be summarized with descriptive statistics for continuous end points and frequency/percentage for categorical end points. [Display omitted] DisclosuresConnor: Astellas Pharma Inc.: Research Funding. Fan: Astellas Pharma Global Development: Current Employment. Gill: Astellas Pharma Global Development: Current Employment. Hill: Astellas Pharma Global Development: Current Employment; Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder. Philipose: Astellas Pharma Global Development: Current Employment. Delgado: Astellas Pharma Global Development: Current Employment. Tiu: Astellas Pharma, Inc.: Current Employment. Reinhardt: Abbvie: Other: Advisory board; Eusa: Other: Advisory board; Astellas Pharma Inc.: Research Funding; Janssen: Other: Advisory board; BluebirdBio: Other: Advisory board; BMS: Other: Advisory board; Novartis: Other: Advisory board; JAZZ: Other: Advisory board. OffLabel Disclosure:New Indication

Assessment of BCOR Internal Tandem Duplications in Pediatric Cancers by Targeted RNA Sequencing

Alterations in the BCOR gene, including internal tandem duplications (ITDs) of exon 15 have emerged as important oncogenic changes that define several diagnostic entities. In pediatric cancers, BCOR ITDs have recurrently been described in clear cell sarcoma of kidney (CCSK), primitive myxoid mesenchymal tumor of infancy (PMMTI), and central nervous system high-grade neuroepithelial tumor with BCOR ITD in exon 15 (HGNET-BCOR ITDex15). In adults, BCOR ITDs are also reported in endometrial and other sarcomas. The utility of multiplex targeted RNA sequencing for the identification of BCOR ITD in pediatric cancers was investigated. All available archival cases of CCSK, PMMTI, and HGNET-BCOR ITDex15 were collected. Each case underwent anchored multiplex PCR library preparation with a custom-designed panel, with BCOR targeted for both fusions and ITDs. BCOR ITD was detected in all cases across three histologic subtypes using the RNA panel, with no other fusions identified in any of the cases. All BCOR ITDs occurred in the final exon, within 16 codons from the stop sequence. Multiplex targeted RNA sequencing from formalin-fixed, paraffin-embedded tissue is successful at identifying BCOR internal tandem duplications. This analysis supports the use of anchored multiplex PCR targeted RNA next-generation sequencing panels for identification of BCOR ITDs in pediatric tumors. The use of post-analytic algorithms to improve the detection of BCOR ITD using DNA panels was also explored.

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells.

Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3-mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 (FLT3wt/FLT3mut). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

Dual inhibition of Fes and Flt3 tyrosine kinases potently inhibits Flt3-ITD+ AML cell growth.

Acute myelogenous leukemia (AML) is often associated with activating mutations in the receptor tyrosine kinase, Flt3, including internal tandem duplications (ITDs) within the regulatory juxtamembrane region. Previous studies have linked Flt3-ITD to the activation of the Fes protein tyrosine kinase in AML, and RNAi-knockdown studies suggest that Fes may be required for Flt3 function. In this study, we tested Fes inhibitors from three different chemical classes for their growth-suppressive activity against Flt3-ITD+ myeloid leukemia cell lines (MV4-11, MOLM-13 and MOLM-14) vs. myeloid cells with wild-type Flt3 (THP-1). All Fes inhibitors selectively inhibited the growth of Flt3-ITD+ AML cells, with IC50 values for diaminopyrimidine and pyrrolopyridine inhibitors ranging from 19 to 166 nM. In contrast, a pyrazolopyrimidine inhibitor was less potent in Flt3-ITD+ AML cells, with IC50 values in the 1.0 μM range. In vitro kinase assays showed that the most potent inhibitors of Flt3-ITD+ AML cell proliferation blocked both Fes and Flt3-ITD kinase activity, while the pyrazolopyrimidine was more selective for Fes vs. Flt3-ITD. All three inhibitors induced significant apoptosis in Flt3-ITD+ AML cells, with potency equivalent to or greater than the established Flt3-ITD inhibitor, tandutinib. Transformation of TF-1 cells with Flt3-ITD resulted in constitutive activation of endogenous Fes, and rendered the cells highly sensitive to all three Fes inhibitors with IC50 values in the 30–500 nM range. The pyrrolopyridine compound also induced apoptotic responses in patient-derived Flt3-ITD+ AML bone marrow cells but not in normal bone marrow mononuclear cells. These results demonstrate that Fes kinase activity contributes to Flt3-ITD signaling in AML, and suggests that dual inhibition of both Flt3 and Fes may provide a therapeutic advantage for the treatment of Flt3-ITD+ AML.

FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.

The receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3), involved in regulating survival, proliferation, and differentiation of hematopoietic stem/progenitor cells, is expressed on acute myeloid leukemia (AML) cells in most patients. Mutations of FLT3 resulting in constitutive signaling are common in AML, including internal tandem duplication (ITD) in the juxtamembrane domain in 25% of patients and point mutations in the tyrosine kinase domain in 5%. Patients with AML with FLT3-ITD have a high relapse rate and short relapse-free and overall survival after chemotherapy and after transplant. A number of inhibitors of FLT3 signaling have been identified and are in clinical trials, both alone and with chemotherapy, with the goal of improving clinical outcomes in patients with AML with FLT3 mutations. While inhibitor monotherapy produces clinical responses, they are usually incomplete and transient, and resistance develops rapidly. Diverse combination therapies have been suggested to potentiate the efficacy of FLT3 inhibitors and to prevent development of resistance or overcome resistance. Combinations with epigenetic therapies, proteasome inhibitors, downstream kinase inhibitors, phosphatase activators, and other drugs that alter signaling are being explored. This review summarizes the current status of translational and clinical research on FLT3 inhibitors in AML, and discusses novel combination approaches. Mol Cancer Ther; 16(6); 991-1001. ©2017 AACR.

Mechanism-Based Combinatorial Strategy for Overcoming FLT3-Inhibitor Resistance in Dual FLT3 ITD and TKD Point Mutations

One-third of acute myeloid leukemias (AML) harbor fms-like tyrosine kinase 3 (FLT3) gene mutations including internal tandem duplication (ITD) and D835 mutations (1). ITD mutations are associated with very poor prognosis (2). Targeted therapy directed against FLT3-ITD mutations has been shown to induce responses in AML patients, albeit only transiently (3). We and others have reported that secondary point mutations, in addition to ITD mutations and aberrant pro-survival signaling pathways such as MAPK or PI3K, are related to the development of resistance (4). To further address the mechanism of FLT3 inhibitor resistance, we generated several murine AML cell lines by introducing dual ITD and point mutations of the tyrosine kinase domain (TKD) 2 of FLT3. We hypothesized that concomitantly targeting FLT3 and MAPK/PI3K may act as a promising approach for overcoming resistance.Previously we showed that E6201, a dual FLT3/MEK inhibitor, demonstrated encouraging anti-leukemia effectiveness in FLT3-ITD and D835G mutated cell lines (5). However, it was less sensitive in triggering cell death in D835Y and ITD/TKD2 dual mutation cells (e.g. ITD plus F691L, Y842C or D835Y etc.). Further investigations demonstrated that those mutated cells have high basal levels of either phospho-FLT3, -ERK, or -AKT. Interestingly, the phosphorylation levels of these proteins were persistently high during long-term exposure to low doses of E6201 (up to 7 to 11 days). As result, these cells became more resistant to subsequent E6201 treatment compared to naïve cells (EC50s were 0.92 vs 0.57 µM and 3.74 vs 1.08 µM in ITD+691 and D835Y mutated cells, respectively). However, concomitant targeting of FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib (XL765) triggered pronounced synergistic pro-apoptotic effects in these pretreated cells (CIs were 0.83 and 0.16 in pretreated ITD+691 and D835Y mutated cells vs. 0.39 and 0.61 in naïve ITD+691 and D835Y mutated cells, respectively; suggesting that this combinatorial strategy may eliminate leukemic cells which have developed resistance during long-term E6201 monotherapy ..Importantly, this synergistic effect was observed even under physiological hypoxic conditions (CI was 0.06 ± 0.01) and in the presence of mesenchymal stromal cells (MSC) (CI was 0.06 ± 0.04) as well, which are considered hallmark conditions of the bone marrow microenvironment. Since CXCR4-SDF-1a signaling axis is associated with MSC-mediated leukemia cell protection, we further tested the combination regime in the presence of CXCR4 antagonist plerixafor. Results demonstrated massive apoptosis induction in MSC co-culture systems (apoptosis induction was 23.9% vs. 91.8% in the absence vs. presence of plerixafor).To minimize combination regime-induced toxicity, we further tested sequential treatment by E6201 and voxtalisib individually. Treatment with voxtalisib followed by E6201 demonstrated identical efficacy as concomitant voxtalisib/E6201 treatment, suggesting that this sequential approach may be beneficial by reducing putative toxicity and enhancing tolerance of the targeted drugs in clinical FLT3-targeted therapy. Conclusion: targeting FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib has potential of preventing the development of FLT3-inhibitor resistance. DisclosuresNo relevant conflicts of interest to declare.

Anti-tumor activity of TAK-659, a dual inhibitor of SYK and FLT-3 kinases, in AML models.

e14091Background: TAK-659 is a reversible, dual inhibitor of SYK and FLT-3 being developed for oncology indications including Acute Myelogenous Leukemia (AML), a disease driven by SYK and/or FLT-3-mediated signaling. Loss of SYK in AML cell lines has been shown to induce myeloid differentiation. FLT-3 is a Class III receptor tyrosine kinase that is normally expressed only in hematopoietic stem and progenitor cells. However, its expression has been found in the blasts of a majority of patients with AML. Activating mutations of FLT-3, including internal tandem duplication (ITDs) within the juxtamembrane region of FLT-3 and point mutations in the FLT-3 activation loop, are observed in approximately 30% of AML patients. These FLT-3 mutations, associated with early relapse and poor survival, represent a critical prognostic factor for AML. Methods: TAK-659 is an investigational inhibitor of SYK and FLT-3 that is currently being evaluated in a Phase 1b/2 clinical trial of AML patients (NCT02323113). TAK-659 inhi...

Recurrent Mutations in CCND3 Confer Clinical Resistance to FLT3 Inhibitors

Background. Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (KD). In recent years, multiple selective and potent FLT3 inhibitors such as quizartinib, PLX3397, crenolanib and ASP2215 have demonstrated encouraging preliminary clinical activity in FLT3 mutant AML. Both quizartinib (Schiller et al, ASCO 2014) and ASP2215 (Levis et al, ASCO 2015) have reported composite complete remission rates (CRc) as high as ~50% in phase II trials. Despite these high response rates, the majority of patients relapse after initial response (acquired resistance) and a significant proportion of patients also fail to respond at all (primary resistance). On-target resistance due to secondary KD mutations in FLT3 is a common cause of acquired resistance to the clinically active FLT3 inhibitors quizartinib (Smith et al. Nature 2012) and PLX3397 (Smith et al, Cancer Discovery 2015). However, the causes of primary clinical resistance to FLT3 inhibitors have not been characterized. We performed targeted capture based sequencing of sorted pre-treatment blasts from 8 responding (R) and 21 non-responding (NR) patients treated on the phase I/II trial of PLX3397 in FLT3-ITD+ AML. We deeply sequenced the coding regions of 585 genes known to be mutated in hematologic malignancies/solid tumors and identified genes mutated only in NR patients (compared to genes mutated in both R and NR patients).Results. The number of mutations detected in genes other than FLT3 ranged from 2-18 per sample. There was no increase in the number of mutations found in patients with pre-existing hematologic conditions or in NR patients. Surprisingly, one of the most frequently mutated genes observed exclusively in NR patients was CCND3, the gene encoding cyclin D3, which has rarely been reported to be mutated in AML, though it is mutated in 38% of sporadic Burkitt's lymphoma (BL). A total of 4 individual mutations in CCND3 (Q276*, Q280fs, R271fs, and T283A) were identified in 3/21 NR patients (one patient had both Q276* and Q280fs). No CCND3 mutations were found in R patients. The identified mutations were the same mutations commonly found in BL, known to result in a more stable isoform of cyclin D3 and retain sensitivity to CDK4/6 inhibitors (Schmitz et al., Nature 2012). Expression of the Q276* and T283A mutations in FLT3-ITD+ MV4;11 cells conferred resistance to apoptosis induced by several FLT3 inhibitors (PLX3397, AC220 and crenolanib). However, inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells by either the CDK4/6 inhibitor palbociclib or the combined FLT3-CDK4/6 inhibitor AMG925 (FLX925) was unable to restore sensitivity to FLT3 inhibition. Moreover, CCND3 mutant MV4;11 cells demonstrated no increase in Rb phosphorylation, suggesting resistance to FLT3 inhibitors facilitated by CCND3 mutations is not predicated on CDK4/6 activation of Rb-dependent E2F-mediated transcription.Conclusions. We have identified recurrent mutations in CCND3, a gene not previously known to be commonly mutated in AML, as a novel cause of clinical primary resistance to FLT3 inhibitors in AML. This represents the first report of a specific non-FLT3 dependent mechanism of clinical resistance to FLT3 inhibitors. Unlike in BL, the functional effects of CCND3 mutations in FLT3-ITD+ AML do not appear to be mediated via CDK4/6 activity and is therefore unresponsive to CDK4/6 inhibition. The molecular mechanism(s) of CCND3-mediated resistance to FLT3 inhibitors are currently being investigated. DisclosuresSmith:Plexxikon: Research Funding; Astellas: Research Funding. Off Label Use: Investigational use of PLX3397 in AML. Hsu:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.

Constitutively Activating Mutations At the FLT3 Activation Loop Residue D835 Are Associated with Clinical Resistance to AC220

Abstract 674 Background.Activating mutations in FLT3 occur in ∼30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (∼25%) and point mutations in the tyrosine kinase domain (KD), primarily at the activation loop (AL) residue D835 (∼5%). FLT3-ITD and FLT3-AL mutations can co-occur in individual patients. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity in FLT3-ITD+ AML, as evidenced by a composite complete remission rate of 45% in 53 chemotherapy refractory/relapsed patients in an interim analysis of a phase II study (Cortes et al, ASH 2011 abstract #614). While some patients without FLT3 mutations in the AC220 phase I dose escalation study responded, no objective response was observed in a small number of FLT3-AL mutant patients (Cortes et al, ASH 2008 abstract #767). We recently reported that secondary mutations at F691, D835 and Y842 in FLT3-ITD confer in vitro resistance to AC220, and that relapse in 8/8 AML patients who initially responded to AC220 was associated with evolution of secondary KD mutations in the FLT3-ITD+ allele (Smith et al., Nature, 2012). In 6 of 8 patients, relapse was associated with D835 substitutions, making this amino acid position the most commonly substituted residue in FLT3-ITD+ patients at the time of disease relapse. Given that D835 mutations in FLT3-ITD appear to confer a high degree of pre-clinical and clinical resistance to AC220, we hypothesized that D835 mutations in the absence of the ITD might also confer resistance to AC220 and could be associated with clinical relapse, especially in patients harboring a mixture of FLT3-WT, FLT3-ITD+, and FLT3-AL mutant blasts. Results.In vitro binding studies revealed that FLT3-D835V and FLT3-D835Y have decreased affinity for AC220 (Kd=4.8 and 7nM, respectively) compared to native FLT3 (Kd=1.8nM). In proliferation assays of BaF/3 cells, FLT3-D835V/Y mutants demonstrated increased relative AC220 resistance (IC50 24 and 6.8nM) compared to FLT3-ITD (IC50 0.13nM). To sensitively and precisely determine the frequency and phase (ITD+ vs ITD-) of mutations in clinical isolates, we utilized single molecule real-time (SMRT; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the KD of FLT3 ITD+ and ITD- alleles. We analyzed ITD- sequences from pretreatment and relapse samples obtained from the 8 FLT3-ITD-positive AML patients with acquired AC220 resistance reported in our previous analysis. Interestingly, all 6 patients with secondary mutations at D835 in ITD+ alleles at relapse also harbored D835 mutant ITD- alleles. All D835 mutations detected in ITD- alleles were also observed in ITD+ alleles from the same patient sample. In ITD+ alleles, we detected D835Y (n=6), D835V (n=3), and D835F (n=2) (range 2.7–50.6% of ITD+ alleles). In ITD- alleles, we found D835Y (n=6), D835V (n=2), and D835F (n=1) (range 3.8–50% of ITD- alleles). One patient had 2 D835 mutations (D835Y/F) in ITD- alleles at relapse. Although 2 of 8 patients evolved the F691L mutation in ITD+ alleles at relapse, this substitution was not detectable in ITD- sequences in any patient. In 7/8 patients, samples obtained immediately prior to AC220 administration were available for analysis. We were unable to document the presence of AC220-resistant mutations in either ITD+ or ITD- sequences in these pretreatment samples, suggesting that these mutants evolved under the selective pressure of AC220 treatment. We are currently performing single cell RT-PCR to determine if D835 mutations in ITD+ and ITD- alleles occur in distinct leukemic cells, which would suggest a polyclonal blast population at relapse. Conclusions.FLT3-AL mutations at D835 confer resistance to AC220 in vitro. The evolution of D835 substitutions in ITD- alleles in the majority of patients who respond and relapse on AC220 suggests that constitutively activating FLT3-AL mutations at residue D835 can confer acquired clinical resistance to AC220. AML patients with FLT3-D835 mutations may exhibit de novo resistance to AC220 and other FLT3 inhibitors. Our findings predict that the clinical activity of potent FLT3 inhibitors that are tolerant of D835 substitutions will mechanistically involve inhibition of D835 mutants in both the presence and absence of an ITD mutation. Disclosures:Chin:Pacific Biosciences: Employment. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Astellas Pharmaceuticals: Consultancy. Travers:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Equity Ownership. Radich:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Shah:Ariad: Consultancy, Research Funding.

Combination of Crenolanib with Sorafenib Produces Synergistic Pro-Apoptotic Effects in FLT3-ITD-Inhibitor-Resistant Acute Myelogenous Leukemias with FLT3 Mutations

Abstract 3591Activating mutations in the FLT3 gene, including internal tandem duplications (ITDs) and missense point mutations of the tyrosine kinase domain (TKD), are frequently observed in AML patients and confer poor prognosis (1). Targeting FLT3 ITD mutations using the multi-kinase inhibitor Sorafenib (a type II kinase inhibitor, which binds to inactive conformation of a kinase ATP pocket)(2) showed impressive anti-leukemia effects in FLT3-ITD mutated AML in Phase I/II clinical trials (3) However, resistance/relapse develops regularly during prolonged Sorafenib therapy (4), in part through acquired point mutations of TKD domains. We postulated that the conformational change of FLT3 protein resulting from acquired point mutations limits the accessibility of sorafenib and leads to resistance (5, 6). Recently, Crenolanib, a novel PDGFRβ tyrosine kinase inhibitor, showed impressive anti-tumor effects by targeting the active conformation of a kinase ATP pocket of FLT3 protein (a type I kinase inhibitor). Therefore, we hypothesize that targeting different sites of FLT3 protein simultaneously using different types of kinase inhbitors may be effective in overcoming sorafenib resistance. We here report that Crenolanib has anti-leukemic activity in Sorafenib-resistant cells which harbor both ITD and acquired TKD point mutations i, and that its combination with Sorafenib in Sorafenib-resistant cells exerts synergistic pro-apoptotic effects.The anti-leukemic activity of Crenolanib was assessed by measuring cell viability (trypan Blue exclusion) and apoptosis induction (annexin V/propidium iodide staining) in isogenic murine Ba/F3 AML cell lines with stable transfection of human FLT3-ITD mutations, in Sorafenib resistant Ba/F3-ITD-Res cells derived from long-term, low-dose exposure of Ba/F3-ITD to Sorafenib in vitro, which harbor N676D and Y842C mutations, and Sorafenib-resistant cell lines Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/F3-ITD+676/842 which carry ITD and TKD point mutations (N676D, Y842C and N676D/Y842C mutations, respectively). Effects of combinatorial regimen employing Crenolanib and Sorafenib were analyzed using CalcuSyn software (combination index (CI) : CI<1 = synergistic, CI>1 = antagonistic effects).Results show that single agent Crenolanib induced cell growth arrest in leukemia cells Ba/F3-ITD, Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/f3-ITD+842/676, at IC50s of 0.012, 0.012 0.037 and 0.038uM, respectively, and induced apoptosis (EC50s) at 0.17, 0.23, 0.19, and 0.22uM, respectively, after 72 hours of treatment. Western Blot showed that Crenolanib profoundly suppressed phosphorylation levels of FLT3 protein and its downstream targets ERK and AKT and induced cleavage of caspase 3. Sorafenib-resistant cells Baf3-ITD+Res and Baf3-ITD+842/676 (EC50s for Sorafenib were 4.2 ± 1.50 and 6.6 ± 0.53 μM, respectively) were exposed to submicromolar concentrations of Crenolanib and Sorafenib concomitantly for 48 h, resulting in impressive synergistic pro-apoptotic effects (CIs were 0.56 ± 0.12 and 0.36 ± 0.04, respectively), implying high synergistic potency of Type I and Type II FLT3 kinase inhibitors, when given concomitantly. In vivo experiments are in progress.Our findings provide therapeutic rationale for a combinatorial treatment strategy with Crenolanib and Sorafenib of FLT-ITD inhibitor-refractory AML. Disclosures:No relevant conflicts of interest to declare.

Acquired Point Mutations of TKD Are Responsible for Sorafenib Resistance in FLT3-ITD Mutant AML,

Abstract 3505Activating mutations of fms-like tyrosine kinase 3 (FLT3), including Internal tandem duplications (ITD) mutations and point mutations of tyrosine kinase domain (TKD), can be detected in 1/3 of acute myeloid leukemia (AML) patients, and are associated with higher risk of relapse. (1) We reported that sorafenib, by directly targeting mutant FLT3-ITD, exerted anti-leukemia effects in AML with FLT3-ITD mutations in Phase I/II clinical trial and increased the complete remission rate to 100% (CR and CRp) in combination with Idarubicin/cytarabine.(2, 3) However, resistance to sorafenib develops in most AML patients with ITD mutations during prolonged therapy and leads to relapse.(3) Therefore, understanding the mechanisms of sorafenib resistance remains a challenge that may aid approaches to overcoming drug resistance in this dismal prognostic group of AML patients. The present study found that acquired point mutations in the TKD1 and TKD2 domains of the FLT3 gene play a crucial role by elevating levels of phosphorylated FLT3 and its downstream signaling proteins.cDNA-based mutation analysis of the FLT3 gene was performed in blood/bone marrow samples from AML patients who showed sensitivity to sorafenib during the first cycles of therapy in Phase I/II trials and then became resistant. Results showed that 50% (3 of 6 cases) developed single or multiple acquired point mutations of the FLT3 gene, which included mutations of D651G(H), G619C and I687F in TKD1 and/or E858K in TKD2. Further, a murine sorafenib-resistant AML cell line (Ba/F3-ITD-Res) was developed by long-term/low-dose exposure to sorafenib in vitro. Sequence analysis identified acquired point mutations of N676D and Y842C. To further investigate the correlation of these point mutations with drug resistance, selected single point mutations were individually introduced into Ba/F3-ITD cells by lentiviral infection. Apoptosis induction was measured after exposure to sorafenib. Results showed varying degrees of resistance in sublines with different point mutations and their EC50s for apoptosis induction were 0.69, 0.61 and 19 μM in D651G, N676D and Y842C mutation-containing cells, respectively. By comparison, EC50 for Ba/F3-ITD was 0.16 μM. However, cells with multiple concomitant point mutations (such as N676D plus Y842C) displayed impressive resistance (EC50 = 32μM). These results suggest that single mutations in either TKD1 or TKD2 are associated with resistance of sorafenib-induced apoptosis, and that structural alterations of TKD2 are more critical than those of TKD1. Further, concomitant mutations in both TKDs strongly impair response to sorafenib, suggesting a pivotal role for the structural integrity of both TKDs in maintaining sensitivity of FLT3-ITD AML cells to sorafenib.To better understand mechanisms of resistance, relevant biomarkers were investigated by Western blot in these cells with point mutations. The results showed that TKD1 mutations (D651G and/or N676D) increased basal levels of phospho–FLT 3 and its downstream targets phospho–ERK, –Stat5, –AKT and –S6K, suggesting that mutations of TKD1 result in increased FLT3 activation. However, one TKD2 mutation (Y842C) which does not result in increased basal levels of phospho-FLT3, was nevertheless resistant to sorafenib-induced suppression of FLT3 targets, implying that Y842C may interfere with the binding of sorafenib to FLT3 by a conformational shift of the adenosine triphosphate (ATP)–binding pocket, which reduces the accessibility for sorafenib and mediates resistance. Notably, cells with concomitant N676D and Y842C mutations in both TKDs displayed higher basal phosphorylation levels of FLT3 and its downstream targets, resulting in increased resistance to sorafenib-induced suppression of phosphorylation, suggesting that synergistic resistance to sorafenib may result from multiple mutations in by both, TKD1 and TKD2.Our findings provide an improved understanding of acquired FLT3-ITD/TDK point mutations associated with sorafenib resistance in FLT3-ITD-mutant AML, and might be useful in developing agents that have the potential of overcoming resistance of FLT3-ITD inhibitor in AML. Disclosures:Ravandi:Bayer: Research Funding; Onyx: Research Funding.

Effect of FMS-Like Tyrosine Kinase-3 Mutations on Prognosis in Acute Myeloid Leukemia: A Single Institution Experience between 2000 and 2007.

Mutations of the FMS-like tyrosine kinase-3 gene (FLT3), including internal tandem duplication (ITD) of the juxtamembrane domain or activating point mutations of the second tyrosine kinase domain involving codon 835 or 836 are among the most common molecular abnormalities in acute myeloid leukemia (AML). A clear negative impact of FLT3 mutation on survival has been demonstrated in AML patients younger than 60 years; however, this negative effect on prognosis or survival has not been confirmed in older patients. We evaluated 661 patients with AML treated at MD Anderson Cancer center between 2000 and 2007 for FLT3 mutations including 281 patients ≤ 60 years and 380 patients > 60 years. Median age for the entire group was 65 years (range, 17 to 86). A FLT3 mutation was present in 124 patients (18.7%). Patients with FLT3 mutations had a significantly higher WBC (median, 16.5 vs. 4.2, p<0.001) and a higher percentage of bone marrow blasts (median, 62% V/S 40%, p<0.001) at initial presentation. These differences were equally seen in both sexes. Patients with FLT3 mutations were younger than those without mutations (median age 58 vs. 64, p=0.005). Patients with FLT3 mutations were less likely to have an antecedent hematological disease compared to those without mutations (p<0.001). FLT3 mutations were less frequent in patients with core binding factor leukemia [t(8;21), inv(16)]. FLT3 mutations were more common in patients with acute promyelocytic leukemia (APL) than other FAB subgroups (p<0.001). Induction death and complete remission rates were not affected by FLT3 status. When favorable prognosis subgroups [APL, t(8;21), inv(16)] were excluded, patients with FLT3 mutations had a significantly shorter overall survival; this difference was seen in patients younger than 60 (p=0.019), as well as in patients over 60 (p=0.015). We have further confirmed a clear negative impact of FLT3 mutations on survival in patients ≤ 60 years and have demonstrated a negative impact on survival even in patients older than 60 years. FLT3 inhibitors should be considered as a component of therapy in older patients with non-favorable AML and FLT3 mutation.

Sorafenib (BAY 43-9006) Directly Targets FLT3-ITD in Acute Myelogenous Leukemia.

Fms-like tyrosine kinase 3 (FLT3) gene mutations, including internal tandem duplication (ITD) and missense point mutations, are frequent molecular abnormalities in acute myeloid leukemia (AML), which are associated with poor prognosis. The Raf-MEK-Erk pathway is one of the downstream targets activated by FLT3 mutations. Our previous studies have demonstrated that Erk is constitutively phosphorylated in the majority of primary AML samples and is associated with poor prognosis in AML, even in the absence of Flt3 abnormalities (Ricciardi et al., 2005; Kornblau et al., 2001 and 2006). Sorafenib (BAY 43-9006), a small molecule inhibitor of c-Raf, has been shown to be a potent inhibitor of wild-type FLT3 (Wilhelm et al., 2004) in addition to targeting Raf and other kinases. We demonstrated that sorafenib selectively induces growth arrest and apoptosis in FLT3-mutant human and murine cell lines at a 1,000-fold lower concentration (IC50 1–3nM) compared to the cells with FLT3 wild-type. Sorafenib also induced a much more pronounced inhibition of colony formation in FLT3-ITD-harboring primary AML cells (IC50=10 ± 2 nM) compared to FLT3 wild-type AML samples (IC50= 2,680 ± 1.8 μM). In a murine leukemia model bearing GFP-transduced Ba/F3-ITD cells, oral administration of sorafenib, at 10 mg/kg/day for 15 days, prolonged the median survival of the mice to 36.5 days compared to 16 days of vehicle group (p=0.002). Histopathology showed significant hepato-splenomegaly in control mice compared to sorafenib-treated mice, and significant reduction of GFP positive leukemia cells in spleen, liver and bone marrow. In an ongoing Phase I clinical trial, 3 patients (pts) with FLT3-ITD have been treated: the 1st had a dramatic decrease of circulating blasts from 92% to 2% in 7 days after oral administration of sorafenib 200 mg/BID and a decrease in BM blasts (95% to 50%), associated with significant down-regulation of phospho-FLT3. The 2nd pt had clearance of peripheral blood blasts from baseline of 97% to undetectable after 7 days and bone marrow blasts decreased from 96% to 25%. The 3rd pt has had a significant decrease in peripheral blasts from 82% to 45% (absolute blasts 10.57 to 0.81 ×109/L). Mechanistically, sorafenib directly inhibited autophosphorylation on tyrosine residues in ITD mutant, but not in FLT3 wild-type protein in the in vitro kinase assays, and blocked downstream targets of mutant FLT3-ITD such as phospho-Akt, phospho-Stat5, phospho-ERK and Pim-1. Further, sorafenib inhibited expression of the anti-apoptotic Mcl-1 and Bcl-XL proteins and induced pro-apoptotic Bim levels in Ba/F3-ITD but not in Ba/F3-FLT3 cells. These results imply that sorafenib directly targets mutant FLT3-ITD protein and its downstream signaling, which may account for the over 1000-fold increased sensitivity to sorafenib in ITD-mutant-harboring AML cells, the significant abrogation of colony formation in ITD-mutant-harboring primary AML cells, and the dramatic drop in circulating blasts in ITD-harboring AML patient. In conclusion, the identification of FLT3-ITD mutant as a main therapeutic target of sorafenib may provide therapeutic guidance in the ongoing Phase I clinical trials in AML and offer therapeutic benefit to AML patients that are expected to respond poorly to chemotherapy.

Novel Mechanism of Activated FLT3-Associated Leukemogenisis: FLT3 Regulates Wnt Signaling by Phosphorylating β-Catenin and Promoting β-Catenin Nuclear Localization.

Mutations of the gene encoding the FLT3 tyrosine kinase are the most common mutation in AML. Recently Wnt-dependent signaling has been suggested to cooperate with FLT3 in myeloid transformation. In the work presented here we have examined the mechanism of the interaction of FLT3 and the Wnt effector β-catenin in FLT3 leukemia cells. In comparison to cells with wild-type FLT3, cells with activating mutations of FLT3 including internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 and the FLT3 D835Y mutation had elevated levels of tyrosine-phosphorylated β-catenin. Although β-catenin was located mainly in the cytoplasm in WT-FLT3 cell lines, it was primarily nuclear in cells with FLT-3 activating mutations. Treatment of activated FLT3 cells with the FLT3 kinase inhibitor AG1296 decreased tyrosine-phoshorylation of β-catenin and downregulated the level of nuclear β-catenin. Treatment of WT-FLT3 cell lines with FLT3 ligand increased tyrosine-phosphorylation of β-catenin and upregulated its nuclear accumulation. Immunoprecipitation experiments demonstrated that FLT3-ITD binds to β-catenin and treatment with AG1296 decreased interaction between FLT3-ITD and β-catenin. In vitro kinase assays using recombinant FLT3 and β-catenin showed that an active FLT3 kinase phosphorylates tyrosine residues of β-catenin directly and AG1296 decreased the activated FLT3-induced tyrosine-phosphorylation of β-catenin. The data demonstrate that activated FLT3 induces tyrosine phosphorylation of β-catenin and accumulation of nuclear β-catenin. We propose that increased β-catenin nuclear signaling is a potential mechanism of activated FLT-3-associated malignant transformation.