scholarly journals Overcoming Resistance to FLT3 Inhibitors in the Treatment of FLT3-Mutated AML

2020 ◽  
Vol 21 (4) ◽  
pp. 1537 ◽  
Author(s):  
Stephen S.Y. Lam ◽  
Anskar Y.H. Leung
Keyword(s):  
Tandem Duplication ◽  
Induction Treatment ◽  
Poor Clinical Outcome ◽  
Conventional Chemotherapy ◽  
Cellular Mechanisms ◽  
Internal Tandem Duplication ◽  
Low Intensity ◽  
Flt3 Inhibitors ◽  
Unfit Patients ◽  
Leukaemia Relapse

Acute myeloid leukaemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine kinase 3 (FLT3) gene is associated with high risk of relapse and poor clinical outcome upon treatment with conventional chemotherapy. FLT3 inhibitors have been approved for the treatment of this AML subtype but leukaemia relapse remains to be a major cause of treatment failure. Mechanisms of drug resistance have been proposed, including evolution of resistant leukaemic clones; adaptive cellular mechanisms and a protective leukaemic microenvironment. These models have provided important leads that may inform design of clinical trials. Clinically, FLT3 inhibitors in combination with conventional chemotherapy as induction treatment for fit patients; with low-intensity treatment as salvage treatment or induction for unfit patients as well as maintenance treatment with FLT3 inhibitors post HSCT hold promise to improve survival in this AML subtype.

scholarly journals Blocking DNA Damage Repair May Be Involved in Stattic (STAT3 Inhibitor)-Induced FLT3-ITD AML Cell Apoptosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuxuan Luo ◽  
Ying Lu ◽  
Bing Long ◽  
Yansi Lin ◽  
Yanling Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Dna Damage Repair ◽  
Underlying Mechanism ◽  
Internal Tandem Duplication ◽  
Mrna Sequencing ◽  
Damage Repair ◽  
Flt3 Inhibitors ◽  
Flt3 Internal Tandem Duplication

The FMS-like tyrosine kinase 3 (FLT3)- internal tandem duplication (ITD) mutation can be found in approximately 25% of all acute myeloid leukemia (AML) cases and is associated with a poor prognosis. The main treatment for FLT3-ITD-positive AML patients includes genotoxic therapy and FLT3 inhibitors, which are rarely curative. Inhibiting STAT3 activity can improve the sensitivity of solid tumor cells to radiotherapy and chemotherapy. This study aimed to explore whether Stattic (a STAT3 inhibitor) affects FLT3-ITD AML cells and the underlying mechanism. Stattic can inhibit the proliferation, promote apoptosis, arrest cell cycle at G0/G1, and suppress DNA damage repair in MV4-11cells. During the process, through mRNA sequencing, we found that DNA damage repair-related mRNA are also altered during the process. In summary, the mechanism by which Stattic induces apoptosis in MV4-11cells may involve blocking DNA damage repair machineries.

The Serine/Threonine Kinase Pim-1 Stabilizes 130 Kilodalton FLT3 and Promotes Aberrant Signaling through STAT5 in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication: Synergistic Apoptosis Induction by Pim-1 and FLT3 Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 942-942 ◽  
Author(s):  
Yingqiu Xie ◽  
Mehmet Burcu ◽  
Maria R. Baer
Keyword(s):  
Half Life ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Internal Tandem Duplication ◽  
Threonine Kinase ◽  
Flt3 Inhibitors ◽  
Flt3 Internal Tandem Duplication

Abstract Abstract 942 Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) results in FLT3 constitutive activation and aberrant signaling in acute myeloid leukemia (AML) cells. FLT3-ITD is associated with adverse treatment outcome in AML, but FLT3 inhibitors have had limited therapeutic efficacy. The oncogenic serine/threonine kinase Pim-1 is upregulated in AML cells with FLT3-ITD. Pim-1 inhibitors are entering clinical trials, and we sought to characterize the role of Pim-1 and the effects of Pim-1 inhibition in FLT3-ITD cells. Wild-type (WT) FLT3 exists predominantly in a 150 kDa complex glycosylated form. In contrast, FLT3-ITD is partially retained in the endoplasmic reticulum (ER) as a misfolded 130 kDa underglycosylated, or high-mannose, species in association with the ER transmembrane chaperone calnexin. In addition, FLT3-ITD also associates with and is stabilized by the cytosolic chaperone heat shock protein (HSP) 90. FLT3-ITD activates signal transducer and activation of transcription (STAT) 5 and upregulates the STAT5 downstream target Pim-1. FLT3 contains a putative Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that FLT3 might be a Pim-1 substrate. FLT3-ITD cell lines studied included MV4-11, MOLM-14 and transfected Ba/F3-ITD, and FLT3 WT cells included BV173, EOL-1 and transfected Ba/F3-WT. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation at serine 112, and Pim-1 expression, FLT3 expression, phosphorylation and co-immunoprecipitation, and STAT5 phosphorylation and expression by Western blot analysis. Pim-1 knockdown was accomplished by infection with lentivirus containing Pim-1 small hairpin RNA (shRNA) or non-target control, and Pim-1 kinase inhibition by incubation with the Pim-1-selective inhibitor quercetagetin. Pim-1 was found to directly interact with and serine-phosphorylate FLT3 from FLT3-ITD, but not FLT3-WT, cells in vitro. Inhibition of Pim-1 kinase disrupted binding of FLT3 to its chaperones calnexin and HSP90, and resulted in decreased expression and half-life of 130 kDa FLT3 and increased expression and half-life of 150 kDa FLT3. The decrease in expression and half-life of 130 kDa FLT3 was partially abrogated by co-incubation with the proteasome inhibitor MG132. Moreover, the increase in 150 Kda FLT3 was abrogated by co-incubation with the glycosylation inhibitor 2-deoxy-D-glucose. Thus Pim-1 maintains FLT3 as a 130 kDa species by enhancing its binding to its chaperones calnexin and HSP90, protecting it from proteasomal degradation and inhibiting its glycosylation to form 150 kDa FLT3. Inhibition of Pim-1 kinase activity also decreased phosphorylation of FLT3 at tyrosine 591, a docking site for binding of FLT3-ITD, but not FLT3-WT, to STAT5, and decreased both STAT5 phosphorylation and expression of Pim-1 itself. In contrast, Pim-1 inhibition had no effect on FLT3 tyrosine kinase activity nor on expression of Pim-2, another Pim kinase family member implicated in promoting survival of FLT3-ITD cells. Finally, the Pim-1 kinase inhibitor quercetagetin and the FLT3 inhibitor PKC412 had a synergistic effect in inducing apoptosis of Ba/F3-ITD cells: We conclude that Pim-1, which is transcriptionally upregulated through STAT5 in FLT3-ITD cells, serine-phosphorylates FLT3-ITD, thereby maintaining it in an underglycosylated form, and promotes STAT5 signaling, and that inhibition of Pim-1 and of FLT3 is synergistic in inducing apoptosis of FLT3-ITD cells. Thus Pim-1 inhibitors should inhibit aberrant signaling upstream as well as downstream of Pim-1 in FLT3-ITD cells, and have the potential to enhance the therapeutic efficacy of FLT3 inhibitors in patients with AML with FLT3-ITD Disclosures: No relevant conflicts of interest to declare.

scholarly journals Concurrent FLT3 Inhibitor and PP2A Activating Drug Treatment Induces Synergistic Cytotoxicity in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication through Proteasomal Degradation of Pim-1 and c-Myc

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3951-3951
Author(s):  
Mario Scarpa ◽  
Shivani Kapoor ◽  
Danilo Perrotti ◽  
Maria R. Baer
Keyword(s):  
Combination Treatment ◽  
Tandem Duplication ◽  
Retroviral Vectors ◽  
Concurrent Treatment ◽  
Internal Tandem Duplication ◽  
Flt3 Inhibitor ◽  
Synergistic Cytotoxicity ◽  
Flt3 Inhibitors ◽  
Drug Treatments ◽  
Flt3 Internal Tandem Duplication

Abstract Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates. Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-MycSer62, phospho-c-MycThr58, phospho-STAT5Tyr694, STAT5, phospho-PP2ATyr307, PP2A, phospho-BADSer112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors. Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-MycSer62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-MycThr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BADS112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment. Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial. Disclosures No relevant conflicts of interest to declare.

Internal Tandem Duplication Mutations of Flt3 (ITD-Flt3) up-Regulate p21 WAF1/CDKN1 Protein That Negatively Regulates Aberrant Proliferation of Hematopoietic Cells Induced by ITD-Flt3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5037-5037
Author(s):  
Mariko Abe ◽  
Louis M. Pelus ◽  
Takeshi Taketani ◽  
Seiji Yamaguchi ◽  
Seiji Fukuda
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Growth Factor ◽  
Gene Deletion ◽  
Tandem Duplication ◽  
Bone Marrow Cells ◽  
Ectopic Expression ◽  
Wild Type ◽  
Internal Tandem Duplication ◽  
Flt3 Inhibitors

Abstract Abstract 5037 Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) are frequently found in patients with acute myeloid leukemia (AML) and are associated with poor prognosis. However, lack of significant efficacy in patients with AML treated with ITD-Flt3 inhibitors in several clinical trials underscores the need for identification of pathways down-stream of ITD-Flt3 that are distinct from normal hematopoietic cells in order to develop novel therapeutic approaches. We have shown that ITD-Flt3 induces expression of the antiapoptotic protein Survivin and cyclin dependent kinase inhibitor (CDKI) p21WAF1/CDKN1 (p21) and that antagonizing Survivin in primary mouse hematopoietic progenitor cells (HPC) expressing ITD-Flt3 inhibits growth factor-independent proliferation (Fukuda et al. Blood 2009). Similarly, ectopic Survivin increases proliferation of mouse primary HPC and this enhancing effect is absent when p21 is functionally deleted (Fukuda et al. Blood 2004). These previous findings suggest that p21 lies down stream of Survivin and involved in facilitating growth factor independent HPC proliferation mediated by ITD-Flt3 signaling. However, analysis of p21 function in growth factor-independent ITD-Flt3 transduced HPC now indicates that p21 in fact negatively regulates ITD-Flt3 signaling. Flt3 ligand (FL) induced marginal proliferation and p21 protein expression in Ba/F3 cells expressing wild-type Flt3. Expression of ITD-Flt3 significantly enhanced proliferation, cell cycle progression and inhibition of apoptosis coincident with up-regulation of p21 in Ba/F3 cells in the absence of FL or IL-3. In contrast, down-regulation of p27Kip1 by ITD-Flt3 was coincidently observed. In addition, higher levels of p21 were observed in ITD-Flt3+ MV4-11 human acute leukemia cells compared to ITD-Flt3- RS4;11 cells. Treatment of MV4-11 cells and Ba/F3 cells expressing ITD-Flt3 with the ITD-Flt3 inhibitor AG1296 significantly reduced p21 expression. In BaF3 cells, elevated p21 expression induced by ITD-Flt3 was suppressed by selective inhibitors for protein kinase A (H89), MAPK (PD98059), PI3-kinase (LY294002), Akt (Akt inhibitor) and p53 (pifithrin-α), suggesting involvement of these signaling pathways in ITD-Flt3 mediated p21 up-regulation. Although ITD-Flt3 induces p21 expression and growth factor-independent proliferation in Ba/F3 cells, control cells expressing both ectopic wild-type Flt3 and p21 did not proliferate in the absence of growth factors, indicating that p21 is not sufficient to substitute for ITD-Flt3. In Ba/F3 cells expressing ITD-Flt3, p21 knock down by shRNA enhanced growth factor-independent proliferation. Ectopic expression of ITD-Flt3 in CFU-GM from p21+/+ mice resulted in significant growth of growth factor-independent CFU-GM in vitro, which was further accelerated by gene deletion of p21 (170 % increase: P<0.05), while co-expression of p21 with ITD-Flt3 in bone marrow cells from p21+/+ mice dramatically decreased growth factor-independent proliferation of c-kit+, Sca-1+, lin- cells (80±6% reduction: P<0.01). In contrast, Survivin gene deletion significantly reduced growth factor-independent CFU-GM proliferation by 74% (P<0.05). Although ITD-Flt3 up-regulates p21 and Survivin independent of hematopoietic growth factors in primary HPC transformed by ITD-Flt3, p21 negatively regulates aberrant growth factor-independent proliferation. This is in contrast to Survivin, which functions up-stream of p21 in normal HPC proliferation and positively regulates ITD-Flt3 signaling. This suggests that p21 antagonizes Survivin down-stream of ITD-Flt3 signaling but cooperates/synergizes with Survivin to enhance proliferation in normal HPC. Since p21 deficiency has been shown to result in fewer total and S-phase CFU and ectopic expression of p21 in p21 −/− bone marrow cells restores total and cycling CFU, p21 positively regulates cell cycle and proliferation of normal HPC, but negatively regulates growth factor-independent proliferation of CFU induced by ITD-Flt3. Our data also suggests that CDKIs p21 and p27 have divergent function in ITD-Flt3 signaling. Manipulation of p21 signaling based on their functional difference between normal and transformed HPC may represent an alternative therapeutic strategy for hematological malignancies expressing ITD-Flt3 that are refractory to ITD-Flt3 inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response

Blood ◽  
2009 ◽  
Vol 113 (17) ◽  
pp. 3938-3946 ◽  
Author(s):  
Keith W. Pratz ◽  
Jorge Cortes ◽  
Gail J. Roboz ◽  
Niranjan Rao ◽  
Omotayo Arowojolu ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Tandem Duplication ◽  
Kinase Inhibitor ◽  
Phase 1 ◽  
Internal Tandem Duplication ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Targeted Agent ◽  
Insight Into

AbstractInternal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting. The clinical studies are registered on www.clinicaltrials.gov as NCT00346632.

scholarly journals FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1425-1432 ◽  
Author(s):  
Keith W. Pratz ◽  
Takashi Sato ◽  
Kathleen M. Murphy ◽  
Adam Stine ◽  
Trivikram Rajkhowa ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Cytotoxic Effect ◽  
Tandem Duplication ◽  
Clinical Status ◽  
Newly Diagnosed ◽  
Wild Type ◽  
Internal Tandem Duplication ◽  
Induce Cell Death ◽  
Flt3 Inhibitors ◽  
Allelic Burden

AbstractWe examined 6 different FMS-like tyrosine kinase-3 (FLT3) inhibitors (lestaurtinib, midostaurin, AC220, KW-2449, sorafenib, and sunitinib) for potency against mutant and wild-type FLT3, as well as for cytotoxic effect against a series of primary blast samples obtained from patients with acute myeloid leukemia (AML) harboring internal tandem duplication (FLT3/ITD) mutations. We found that inhibition of FLT3 autophosphorylation in a FLT3/ITD specimen does not always induce cell death, suggesting that some FLT3/ITD AML may not be addicted to FLT3 signaling. Relapsed samples and samples with a high mutant allelic burden were more likely to be responsive to cytotoxicity from FLT3 inhibition compared with the samples obtained at diagnosis or those with a low mutant allelic burden. These FLT3 inhibitors varied to a considerable degree in their selectivity for FLT3, and this selectivity influenced the cytotoxic effect. These results have important implications for the potential therapeutic use of FLT3 inhibitors in that patients with newly diagnosed FLT3-mutant AML might be less likely to respond clinically to highly selective FLT3 inhibition.

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