A novel combination regimen of BET and FLT3 inhibition for FLT3-ITD acute myeloid leukemia

Acute myeloid leukemia (AML) patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors improve overall survival, but their efficacy is limited and most patients who relapse will ultimately die of the disease. Even with potent FLT3 inhibition, the disease persists within the bone marrow (BM) microenvironment, mainly due to BM stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as MYC and BCL2, but these drugs thus far have shown only limited single-agent clinical potential. We demonstrate here, using pre-clinical and clinical correlative studies, that the novel 4-azaindole derivative, {"type":"entrez-protein","attrs":{"text":"PLX51107","term_id":"1321741095","term_text":"PLX51107"}}PLX51107, has BET-inhibitory activity in vitro and in vivo. The combination of BET and FLT3 inhibition induces a synergistic anti-leukemic effect in a murine xenograft model of FLT3- ITD AML, and against primary FLT3-ITD AML cells co-cultured with BM stroma. Using suppression of MYC as a surrogate for BET inhibition, we demonstrate BET inhibition in human patients. The short plasma half-life of {"type":"entrez-protein","attrs":{"text":"PLX51107","term_id":"1321741095","term_text":"PLX51107"}}PLX51107 results in intermittent target inhibition to promote tolerability while overcoming the protective effect of the microenvironment. Mechanistically, the synergistic cytotoxicity is associated with suppression of key survival genes such as MYC. These data provide the scientific rationale for a clinical trial of a BET plus FLT3 inhibitor for the treatment of relapsed/refractory FLT3-ITD AML. A clinical trial of {"type":"entrez-protein","attrs":{"text":"PLX51107","term_id":"1321741095","term_text":"PLX51107"}}PLX51107 as monotherapy in patients with different malignancies is underway and will be reported separately.     

Analysis of induction efficacy and prognostic factors in FLT3-ITD positive acute myeloid leukemia in the real world

<正>Objective To investigate the efficacy and prognostic factors of induction therapy in FLT3-ITD+acute myeloid leukemia (AML) in the real world data. Methods From January 2013 to December 2016,114 de novo patients with FLT3-ITD+AML were enrolled in this study. Out of     

FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression

FMS-like tyrosine kinase 3 (FLT3) is almost universally expressed in B-precursor childhood acute lymphoblastic leukemia (ALL). Cases of ALL with MLL gene rearrangements and those with high hyperdiploidy (> 50 chromosomes) express the highest levels of FLT3, and activating mutations of FLT3 occur in 18% of MLL-rearranged and 28% of hyperdiploid ALL cases. We determined the antileukemic activity of CEP-701, a potent and selective FLT3 inhibitor, in 8 ALL cell lines and 39 bone marrow samples obtained at diagnosis from infants and children with various subtypes of ALL. CEP-701 induced pronounced apoptotic responses in a higher percentage of samples that expressed high levels of FLT3 (74%, n = 23) compared with samples with low levels of expression (8%, n = 13; P = .0003). Sensitivity to FLT3 inhibition was particularly high in samples with MLL gene rearrangements (82%, n = 11; P = .0005), high hyperdiploidy (100%, n = 5; P = .0007), and/or FLT3 mutations (100%, n = 4; P = .0021). Seven of 7 sensitive samples examined by immunoblotting demonstrated constitutively phosphorylated FLT3 that was potently inhibited by CEP-701, whereas 0 of 6 resistant samples expressed constitutively phosphorylated FLT3. We conclude that the FLT3 inhibitor CEP-701 effectively suppresses FLT3-driven leukemic cell survival. Clinical testing of CEP-701 as a novel molecularly targeted agent for the treatment of childhood ALL is warranted.     

Improved clinical outcome of acute myeloid leukemia with FLT3-ITD mutation treated with sorafenib

<正>Objective To analyze the efficacy of sorafenib on the treatment of patients diagnosed as acute myeloid leukemia(AML)with FLT3-ITD mutation.Methods From January 2012 to February 2015,42 cases of AML with FLT3-ITD mutation according to MICM(morphology,immunology,cytogenetics and molecular)diagnosis system in our hospital were retrospectively analyzed.Thirty-two     

Sorafenib in combination with chemotherapy as first-line therapy for FLT3-ITD positive acute myeloid leukemia

正Objective To analyze the clinical features of acute myeloid leukemia patients with Fms-like tyrosine kinase3 interal tandem duplication(FLT3-ITD)mutation and the therapeutic effect of sorafenib in combination with chemotherapy as first-line therapy for these patients.Methods Clinical features and therapeutic effect were te-     

FLT3 inhibitors in acute myeloid leukemia

The fms-like tyrosine kinase 3 (FLT3) plays an important role in both normal and malignant hematopoiesis. Activating mutations in the FLT3 receptor can be detected in approximately 30% of acute myeloid leukemias (AMLs) and are associated with a distinctly poor clinical outcome for patients. There are now several classes of FLT3 inhibitors in development with varying degrees of potency and selectivity for the target, including several in late-phase clinical trials in combination with chemotherapy. Major clinical responses in AML patients receiving single-agent FLT3 inhibitors have been rare, although transient peripheral blood blast reduction is common. Given such biological suggestion and preclinical activity, FLT3 inhibitors hold promise in improving the outcome of patients with mutant FLT3 AML. This review summarizes the current attempts to target this molecule, with emphasis on the validity of the target, the results of the clinical trials evaluating the FLT3 inhibitors in AML, the optimal use of these compounds and the mechanisms of resistance.     

Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models

FLT3 internal tandem duplication (FLT3-ITD) mutations account for approximately 25% of adult acute myeloid leukemia (AML) cases and are associated with poor prognosis. Venetoclax, a selective BCL-2 inhibitor, has limited monotherapy activity in relapsed/refractory AML with no responses observed in a small subset of FLT3-ITD+ patients. Further, FLT3-ITD mutations emerged at relapse following venetoclax monotherapy and combination therapy suggesting a potential mechanism of resistance. Therefore, we investigated the convergence of FLT3-ITD signaling on the BCL-2 family proteins and determined combination activity of venetoclax and FLT3-ITD inhibition in preclinical models. In vivo, venetoclax combined with quizartinib, a potent FLT3 inhibitor, showed greater anti-tumor efficacy and prolonged survival compared to monotherapies. In a patient-derived FLT3-ITD+ xenograft model, cotreatment with venetoclax and quizartinib at clinically relevant doses had greater anti-tumor activity in the tumor microenvironment compared to quizartinib or venetoclax alone. Use of selective BCL-2 family inhibitors further identified a role for BCL-2, BCL-XL and MCL-1 in mediating survival in FLT3-ITD+ cells in vivo and highlighted the need to target all three proteins for greatest anti-tumor activity. Assessment of these combinations in vitro revealed synergistic combination activity for quizartinib and venetoclax but not for quizartinib combined with BCL-XL or MCL-1 inhibition. FLT3-ITD inhibition was shown to indirectly target both BCL-XL and MCL-1 through modulation of protein expression, thereby priming cells toward BCL-2 dependence for survival. These data demonstrate that FLT3-ITD inhibition combined with venetoclax has impressive anti-tumor activity in FLT3-ITD+ AML preclinical models and provides strong mechanistic rational for clinical studies.     

Mechanisms of resistance against PKC412 in resistant FLT3-ITD positive human acute myeloid leukemia cells

Treatment of acute myeloid leukemia (AML) remains challenging with many patients harboring unfavorable prognostic parameters such as FLT3 internal tandem duplication (FLT3-ITD) mutations leading to a constitutively activated FLT3-receptor tyrosine kinase (RTK). Activation of proteins by phosphorylation of tyrosine residues is a common mechanism in leukemia development. Therefore, specific tyrosine kinase inhibitors (TKI) have been developed for AML therapy and are currently under investigation. The staurosporine derivate PKC412 (Midostaurin) was found to be an effective inhibitor of the FLT3-RTK and is currently undergoing clinical trials for FLT3-mutated AML patients. Since resistance towards TKIs has been observed in vitro and in clinical trials, we have generated a PKC412-resistant clone (MV4-11r) of the human myelomonoblastic cell line MV4-11, which carries a homozygous FLT3-ITD mutation. MV4-11r displayed higher vitality after addition of PKC412 compared with MV4-11 with a pronounced reduction of apoptotic cells. Cytogenetic characterization revealed the acquisition of additional aberrations in the resistant cell line such as clonal alterations at chromosome 13q with additional FLT3 signals. Microarray analysis revealed significant expression changes in several genes prior to and after incubation with PKC412. The expression status of candidate genes being regulated by FLT-ITD like JAG1, p53, MCL-1, C-KIT, and FLT3/-L was confirmed by real-time PCR. In summary, resistance against PKC412 appears to be mediated by up-regulation of anti-apoptotic genes and down-regulation of proapoptotic signals as well as genes that are involved in normal and malignant hematopoiesis.     

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFbeta-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFbeta-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFbeta-SMMHC/FLT3-ITD-expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFbeta-SMMHC-expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFbeta-SMMHC/FLT3-ITD-reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFbeta-SMMHC in an animal model of inv(16)-associated AML.     

Potential targeting of FLT3 acute myeloid leukemia

Aberrant FLT3 receptor signaling is common in acute myeloid leukemia (AML) and has important implications for the biology and clinical management of the disease. Patients with FLT3-mutated AML frequently present with critical illness, are more likely to relapse after treatment, and have worse clinical outcomes than their FLT3 wildtype counterparts. The clinical management of FLT3-mutated AML has been transformed by the development of FLT3 inhibitors, which are now in use in the frontline and relapsed/refractory settings. However, many questions regarding the optimal approach to the treatment of these patients remain. In this paper, we will review the rationale for targeting the FLT3 receptor in AML, the impact of FLT3 mutation on patient prognosis, the current standard of care approaches to FLT3-mutated AML management, and the diverse array of FLT3 inhibitors in use and under investigation. We will also explore new opportunities and strategies for targeting the FLT3 receptor. These include targeting the receptor in patients with non-canonical FLT3 mutations or wild-type FLT3, pairing FLT3 inhibitors with other novel therapies, using minimal residual disease testing to guide the targeting of FLT3, and novel immunotherapeutic approaches.     

FLT3 mutations in childhood acute lymphoblastic leukemia

Activating mutations of the FLT3 receptor tyrosine kinase are common in acute myelogenous leukemia (AML) but are rare in adult acute lymphoblastic leukemia (ALL). We have recently shown that FLT3 is highly expressed and often mutated in ALLs with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Because hyperdiploid ALL samples also show high-level expression of FLT3, we searched for the presence of FLT3 mutations in leukemic blasts from 71 patients with ALL. The data show that approximately 25% (6 of 25) of hyperdiploid ALL samples possess FLT3 mutations, whereas only 1 of 29 TEL/AML1-rearranged samples harbored mutations (P =.04, Fisher exact test). Three mutations are novel in-frame deletions within a 7-amino acid region of the receptor juxtamembrane domain. Finally, 3 samples from patients whose disease would relapse harbored FLT3 mutations. These data suggest that patients with hyperdiploid or relapsed ALL might be considered candidates for therapy with newly described small-molecule FLT3 inhibitors.     

异基因造血干细胞移植治疗Fms样酪氨酸激酶3基因内部串联重复(FLT3-ITD)阳性的急性髓性白血病的疗效分析

 目的 分析异基因造血干细胞移植(allo-HSCT)治疗伴有Fms样酪氨酸激酶3基因内部串联重复(FLT3-ITD)阳性的急性髓性白血病(AML)的疗效,探讨不同移植方式及疾病状态对该类患者预后的影响。方法 2006年10月至2012年10月在苏州大学附属第一医院行allo-HSCT的AML患者共314例,其中FLT3-ITD阳性54例,回顾性分析allo-HSCT对FLT3-ITD阳性AML患者的临床疗效。结果 54例FLT3-ITD阳性患者3年总生存(OS)率为56%, 3年无白血病生存(LFS)率为47%。其中同胞人类白细胞抗原(HLA)全相合及亲缘HLA单倍型相合造血干细胞移植的患者3年OS率分别为56%和60%,3年LFS率为45%和54%。两组在OS时间及LFS时间方面的差异均无统计学意义(χ²=0.074,P=0.786; χ²=0.006,P=0.941)。47例(87.0%)患者移植前本病处于首次完全缓解(CR1),7例(13.0%)患者移植前本病处于非CR1期。处于CR1期的患者的生存显著优于非CR1期患者。结论 allo-HSCT是FLT3-ITD阳性AML患者的有效治疗方法,亲缘HLA单倍型相合造血干细胞移植与同胞HLA全相合移植疗效相似。疾病复发是影响其疗效的主要因素。
        

Subclinical minute FLT3-ITD clone can be detected in clinically FLT3-ITD-negative acute myeloid leukaemia at diagnosis

Recent advances in next-generation sequencing (NGS) have enabled the detection of subclinical minute FLT3-ITD. We selected 74 newly diagnosed, cytogenetically normal acute myeloid leukaemia (AML) samples in which FLT3-ITD was not detected by gel electrophoresis. We sequenced them using NGS and found minute FLT3-ITDs in 19 cases. We compared cases with clinically relevant FLT3-ITD (n = 37), cases with minute FLT3-ITD (n = 19) and cases without detectable FLT3-ITD (n = 55). Molecular characteristics (location and length) of minute FLT3-ITD were similar to those of clinically relevant FLT3-ITD. Survival of cases with minute FLT3-ITD was similar to that of cases without detectable FLT3-ITD, whereas the relapse rate within 1 year after onset was significantly higher in cases with minute FLT3-ITD. We followed 18 relapsed samples of cases with clinically FLT3-ITD-negative at diagnosis. Two of 3 cases with minute FLT3-ITD relapsed with progression to clinically relevant FLT3-ITD. Two of 15 cases in which FLT3-ITD was not detected by NGS relapsed with the emergence of minute FLT3-ITD, and one of them showed progression to clinically relevant FLT3-ITD at the second relapse. We revealed the clonal dynamics of subclinical minute FLT3-ITD in clinically FLT3-ITD-negative AML. Minute FLT3-ITD at the initial AML can expand to become a dominant clone at relapse.     

Potent co-operation between the NUP98-NSD1 fusion and the FLT3-ITD mutation in acute myeloid leukemia induction

The NUP98-NSD1 fusion, product of the t(5;11)(q35;p15.5) chromosomal translocation, is one of the most prevalent genetic alterations in cytogenetically normal pediatric acute myeloid leukemias and is associated with poor prognosis. Co-existence of an FLT3-ITD activating mutation has been found in more than 70% of NUP98-NSD1-positive patients. To address functional synergism, we determined the transforming potential of retrovirally expressed NUP98-NSD1 and FLT3-ITD in the mouse. Expression of NUP98-NSD1 provided mouse strain-dependent, aberrant self-renewal potential to bone marrow progenitor cells. Co-expression of FLT3-ITD increased proliferation and maintained self-renewal in vitro. Transplantation of immortalized progenitors co-expressing NUP98-NSD1 and FLT3-ITD into mice resulted in acute myeloid leukemia after a short latency. In contrast, neither NUP98-NSD1 nor FLT3-ITD single transduced cells were able to initiate leukemia. Interestingly, as reported for patients carrying NUP98-NSD1, an increased Flt3-ITD to wild-type Flt3 mRNA expression ratio with increased FLT3-signaling was associated with rapidly induced disease. In contrast, there was no difference in the expression levels of the NUP98-NSD1 fusion or its proposed targets HoxA5, HoxA7, HoxA9 or HoxA10 between animals with different latencies to develop disease. Finally, leukemic cells co-expressing NUP98-NSD1 and FLT3-ITD were very sensitive to a small molecule FLT3 inhibitor, which underlines the significance of aberrant FLT3 signaling for NUP98-NSD1-positive leukemias and suggests new therapeutic approaches that could potentially improve patient outcome.