scholarly journals Momelotinib is a highly potent inhibitor of FLT3-mutant AML

2021 ◽  
Author(s):  
Mohammad Azhar ◽  
Zachary Kincaid ◽  
Meenu Kesarwani ◽  
Arhama Ahmed ◽  
Mark Wunderlich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kinase Inhibitor ◽  
Leukemic Cells ◽  
Activation Loop ◽  
Growth Factor Signaling ◽  
Durable Response ◽  
Allogenic Stem Cell Transplantation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogenic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues, D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL) and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

scholarly journals Momelotinib is a highly potent inhibitor of FLT3-mutant AML

2021 ◽  
Author(s):  
Mohammad Azhar ◽  
Zachary Kincaid ◽  
Meenu Kesarwani ◽  
Tahir Latif ◽  
Daniel Starczynowski ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kinase Inhibitor ◽  
Initial Response ◽  
Leukemic Cells ◽  
Activation Loop ◽  
Growth Factor Signaling ◽  
Durable Response ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogeneic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL), and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

scholarly journals Momelotinib Is a Highly Potent Inhibitor of FLT3-Mutant AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 206-206
Author(s):  
Mohammad Azhar ◽  
Zachary Kincaid ◽  
Meenu Kesarwani ◽  
Mark Wunderlich ◽  
Tahir Latif ◽  
...  
Keyword(s):  
Overall Survival ◽  
Growth Factor ◽  
Activation Loop ◽  
Type I ◽  
Growth Factor Signaling ◽  
Intrinsic Resistance ◽  
Durable Response ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Jak2 Inhibition

Abstract Approximately one-third of AML patients harbor kinase activating mutations in FLT3. Several small-molecule first generation FLT3 tyrosine kinase inhibitors (TKIs) have been evaluated in the last two decades, but none could induce a durable response possibly due to poor pharmacokinetics and target selectivity. Second generation FLT3 inhibitors such as, quizartinib, gilteritinib, and crenolanib, were designed for greater selectivity with a narrow kinome-profile and better pharmacokinetic properties, but they failed to induce a durable response. Possibly due to intrinsic resistance conferred by growth factor signaling in the bone marrow resident leukemic cells, which serve as a reservoir to develop resistance resulting to disease relapse. Activation of Jak2 signaling by chemokines and cytokines from the stroma have been reported to confer TKI refractoriness. Recent studies from Martin Carroll's group showed that both GMCSF and IL-3 confer resistance by activating JAK2 signaling, which can be suppressed by combined FLT3 and JAK2 inhibition. However, so far, it is not established whether upfront FLT3/Jak2 inhibition will provide durable response. For instance, AML patients who achieved complete remission lacking FLT3-ITD clones showed better overall survival than patients with measurable MRD, suggesting that eradicating the FLT3-ITD clones will have a deeper response with better overall survival. Thus, an ideal FLT3 inhibitor should be able to suppress FLT3-ITD resistant mutants, and growth factor activated JAK2 signaling while sparing c-KIT receptor and hERG to avoid myelosuppression and cardiotoxicity, respectively. Here we show that Jak2 inhibitor, momelotinib, is an equipotent type-I FLT3 inhibitor (Fig 1A-D). Biochemical and structural modeling revealed that it binds to an active conformation of FLT3 kinase. Therefore, like gilteritinib, it efficiently suppresses the resistance conferred by activation loop mutations (Fig1 E-H). Moreover, its lack of activity against c-KIT and inhibition of ACVR1 provides additional benefit in alleviating myelosuppression and anemia, which is commonly observed with currently used JAK2 and FLT3 inhibitors (ruxolitinib, fedratinib, and quizartinib). Perhaps more interestingly, momelotinib efficiently suppressed the disease in a preclinical model of AML using NSGS mice which recapitulates cytokine induce refractoriness as usually observed in clinical setting (Fig1 I-L). Our preclinical data provide evidence that momelotinib is an equipotent dual JAK2/FLT3 inhibitor and suppresses resistance conferred by both activation-loop mutations and growth-factor signaling. These data provide evidence that momelotinib treatment will have clinical activity in FLT3-mutated AML. Thus, warrants its clinical evaluation. Figure 1 Figure 1. Disclosures Starczynowski: kurome Inc: Consultancy.

scholarly journals A Mixed Type 1 and Type 2 Kinase Inhibitor That Overrides FLT3 F691 and D835 Resistance Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1074-1074 ◽  
Author(s):  
Jack Lin ◽  
Ying Zhang ◽  
Bernice Matusow ◽  
Adam Mumy ◽  
Garson Tsang ◽  
...  
Keyword(s):  
Mixed Type ◽  
Kinase Inhibitor ◽  
Binding Mode ◽  
Resistance Mutations ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Introduction: Mutations in FLT3, a class III receptor tyrosine kinase predominantly expressed on hematopoietic progenitor cells, represent the most common genetic alteration in patients with acute myeloid leukemia (AML). Approximately 25% of patients with AML harbor internal tandem duplications (ITD) within the juxtamembrane domain of FLT3. Quizartinib, the first selective FLT3 tyrosine kinase inhibitor (TKI) to show clinical activity, frequently achieves a composite complete remissions (CRc) rate of approximately 50% in relapsed and refractory FLT3-ITD+AML patients. However, patient samples obtained at the time of relapse with quizartinib revealed resistance-conferring FLT3 mutations at two kinase domain residues (F691L and D835V/Y/F). The crystal structure of the FLT3-quizartinib complex indicates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and with F830 within the DFG motif in the activation loop (A-loop). This reliance makes quizartinib responses critically vulnerable to gatekeeper (F691) and A-loop (D835) substitutions. This has led to the identification of TKIs that have activity against quizartinib-resistant FLT3 mutants, including type 1 inhibitors (e.g. crenolanib, ASP2215) targeting specifically D835 mutants, and type 2 inhibitors (e.g. pexidartinib, ponatinib) active against F691L. Until now, no FLT3 inhibitor has demonstrated potent inhibition of both classes of resistance mutations. Methods: To identify FLT3 inhibitors with activity against both gatekeeper and A-loop mutants, we used a modular approach to systematically alter the constituents of quizartinib to optimize interactions with the mutant proteins guided by X-ray structures and modeling. The compounds were profiled through a panel of in vitro growth assays and in vivo studies using Ba/F3 cells engineered to express FLT3-ITD/F691L and FLT3-ITD/D835Y, and MOLM14 quizartinib-resistant clones (MOLM14/F691L and MOLM14/D835Y) that have acquired the same mutations after chronic exposure to quizartinib. When injected into the tail vein of nude mice, the engineered Ba/F3 cells home to the spleen and proliferate, causing marked splenomegaly, whereas MOLM14 parental cells and MOLM14 resistant clones significantly reduce the overall survival of mice. Ba/F3-induced splenomegaly is directly dependent on the activity of the expressed mutant FLT3 and inhibition of splenomegaly measures target engagement in vivo. Analysis of efficacy in the MOLM14 survival models was done by Kaplan-Meier estimates and the logrank statistic. Results: Structure-guided lead optimization generated a series of FLT3 inhibitors with a mixed type 1 and type 2 binding mode and equal potency against the two classes of resistant mutants. Further optimization to improve pharmaceutical properties and maximize in vivo efficacy led to development candidate PLX3623. PLX3623 potently inhibited the growth of two Ba/F3 cell lines expressing F691L and D835Y mutants, with IC50 values of 0.18 (±0.04) and 0.15 (±0.03) nM, respectively. PLX3623 had a comparable antiproliferative effect on the MOLM14 resistant clones, with IC50values of 0.52 (±0.14) and 0.26 (±0.08) nM for the F691L and D835Y clones, respectively. In vivo PLX3623 treatment resulted in dose-dependent reduction in Ba/F3-induced splenomegaly, achieving >90% inhibition at 3 mg/kg dose. PLX3623 at 3mg/kg increased the length of survival of MOLM14 parental cell recipients by 120%, compared to the 80% increase afforded by 3 mg/kg quizartinib. At 3mg/kg dose, PLX3623 extended the survival of mice that received the two MOLM14 resistant clones by 80%; quizartinib at the same dose provided no survival benefit. Conclusion: Clonal evolution and tumor heterogeneity present a major challenge to targeted therapy and personalized cancer medicine. While acquired mutations individually may be actionable targets, often multiple inhibitors are required to combat the clonal diversity in a refractory setting. PLX3623, a FLT3 inhibitor with a mixed type 1 and type 2 binding mode, is the first TKI that is active against mutations conferring resistance through independent structural mechanisms. The in vitro and in vivo potency and favorable nonclinical safety profile of PLX3623 support its clinical development as a potentially best-in-class therapy to overcome clinical resistance to first generation FLT3 inhibitors. Disclosures Lin: Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Matusow:Plexxikon Inc.: Employment. Mumy:Plexxikon Inc.: Employment. Tsang:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Powers:Plexxikon Inc.: Employment. Spevak:Plexxikon Inc.: Employment. Severson:Plexxikon Inc.: Employment. Tsai:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment.

scholarly journals p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120774 ◽  
Author(s):  
Miyuki Inoue-Mochita ◽  
Toshihiro Inoue ◽  
Tomokazu Fujimoto ◽  
Takanori Kameda ◽  
Nanako Awai-Kasaoka ◽  
...  
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Trabecular Meshwork ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
P38 Map Kinase ◽  
Collagen Production ◽  
Trabecular Meshwork Cells ◽  
Transforming Growth Factor Β2

p53 Activation of Mesenchymal Stromal Cells (MSC) Partially Abrogates Microenvironment-Mediated Resistance to FLT3 Inhibition In FLT3/ITD AML

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2151-2151
Author(s):  
Kensuke Kojima ◽  
Teresa McQueen ◽  
Elizabeth J. Shpall ◽  
Rui-Yu Wang ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Protective Effect ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Protein Levels ◽  
Dismal Prognosis ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
P53 Activation

Abstract Abstract 2151 Fms-like tyrosine kinase-3 (FLT3) inhibitors have recently been introduced to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3/ITD mutations. However, while ciculating blasts are rapidly eliminated, bone marrow (BM) responses are in general less impressive (Zhang et al., JCI 2009). One potential explanation for the reduced bone marrow response compared to the striking activity against circulating blast cells may be microenvironmental resistance to FLT3 inhibitors, including protection of FLT3/ITD+ blasts through the SDF-1 (CXCL12)/CXCR4 axis (Zeng et al., BLOOD 2009) and novel strategies to overcome microenvironmental resistance may further enhance the clinical benefit of FLT3 inhibitors. Here we investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3/ITD AML. We confirmed that Mdm2-p53 interaction and mutant FLT3 signaling in leukemic blasts were inhibited by the Mdm2 inhibitor Nutlin-3a and the selective FLT3 inhibitor FI-700, respectively, as previously reported (Kojima et al., BLOOD 2004; Kojima et al., LEUKEMIA 2009). BM samples were obtained from normal individuals or from FLT3/ITD AML patients with more than 70% leukemia cells after informed consent. BM-derived stromal cells were seeded in 12-well plates in MEM-alpha medium and exposed to Nutlin-3a for 24 hours, gamma-irradiated (2 Gy), or treated with 100 nM doxorubicin. The wells were then washed three times and AML cells were added. Cell cycle distribution was determined by propidium iodide staining and apoptosis by Annexin V binding. In FLT3/ITD AML cell lines and primary cells, apoptosis was induced by FI-700, but apoptosis induction was diminished under stromal coculture conditions (43.2 ± 0.5% versus 17.0 ± 1.2% in MOLM-13; p < .01). FI-700–induced reduction of Mcl-1 protein levels and activation of Bax were essentially abrogated when FLT3/ITD AML cells were co-cultured with MSC (20.9 ± 1.6% versus 8.3 ± 0.3% cells with activated Bax levels in MOLM-13;p < .01). Separation of MOLM-13 cells from stroma by 0.4 μM culture filters did not affect the protective effect of stromal cells (21.2 ± 2.3% versus 21.6 ± 1.6%). The protection appeared to be mediated partially by SDF-1/CXCR4 signaling because the addition of recombinant SDF-1 to control medium protected FLT3/ITD AML cells from FI-700–induced apoptosis while the addition of anti-CXCR4 antibody (R&D Systems) partially abrogated the protection conferred by stromal cells. The protective effect of stromal cells on MOLM-13 was significantly reduced when MSC were exposed to p53 inducers including Nutlin-3a (17.0 ± 1.2% versus 26.0 ± 1.8%; p < .01), gamma-irradiation (13.5 ± 3.0% versus 18.6 ± 3.1%; p < .05) or doxorubicin (14.2 ± 3.0% versus 19.7 ± 0.9%; p < .05), although p53 activation was not itself cytotoxic to stromal cells. However, SDF-1 mRNA and protein levels were reduced in p53-activated stroma cells. Our findings confirm that selective FLT3 inhibition may not eradicate FLT3/ITD AML cells protected by stroma and show, for the first time, that p53 activation in stroma cells blunts stroma cell-mediated resistance to a kinase, here a FLT3 inhibitor, in part through regulation of SDF-1. Results suggest that combinations of HDM2/p53 (R7112/Nutlin-3a, Andreeff et al. ASH 2010) and/or CXCR4 inhibitors (Plerixafor, Konopleva et al. ASH 2010) with FLT3 inhibitors should be investigated in clinical trials targeting FLT3 mutant leukemias, with the dual goal of inducing apoptosis in leukemic cells and, concomittantly, reduce the protective effects exerted by the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Responsiveness of FLT3-ITD+ AML to Kinase Inhibition Correlates with High Allele Ratio and Relapsed Disease but Not to Inhibition of Canonical Targets

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2371-2371
Author(s):  
Mayumi Sugita ◽  
Grace R Jeschke ◽  
Martin P. Carroll ◽  
Alexander E. Perl
Keyword(s):  
Signal Transduction ◽  
Clinical Response ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Liquid Culture ◽  
Kinase Inhibitor ◽  
Signal Transduction Pathways ◽  
Allele Ratio ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase and is the most commonly mutated gene in acute myeloid leukemia (AML). Second generation FLT3 inhibitors such as quizartinib (AC220) are clinically active in relapsed FLT3-ITD+ patients. However, not all patients respond and to date primary resistance has not been characterized. A previous study proposed that AML cells from patients with relapsed FLT3 mutant AML and samples with high allelic burden for FLT3 ITD are more sensitive to FLT3 inhibitor cytotoxicity (Pratz KW, et al Blood 2010). We performed studies to test this hypothesis and determine if cells with homodimeric FLT3 ITD are more heavily dependent on FLT3 ITD for growth and survival than cells expressing heterodimeric FLT3 WT:FLT3 ITD. 16 primary AML samples that contain FLT3 ITD mutations were incubated in increasing concentrations of the second generation FLT3 inhibitor crenolanib and assayed for survival in short term liquid culture assays. Only 6 samples demonstrated greater than 30% inhibition of survival in this culture system whereas 10 samples showed little or no cytotoxic response. Consistent with previous results (Pratz, et. al, Blood 2010), responding samples tended to be from relapsed patients and to have higher FLT3 ITD allelic ratios. We then analyzed FLT3 expression and phosphorylation levels as well as inhibition of and crenolanib inhibition of FLT3 phosphorylation, as well as the canonical downstream signal transduction pathways STAT5, ras/MAPK, and PI3K/AKT/mTOR in 13 FLT3-ITD+ primary AML samples. For 11/13 samples, crenolanib strongly inhibited phosphorylation of FLT3 kinase. However, neither FLT3 protein expression nor baseline phosphorylation level correlated with cytotoxic response in liquid culture assays. Crenolanib inhibited phosphorylation of STAT5, ribosomal S6 and ERK to varying degrees and inhibition of none of these pathways consistently correlated with cytotoxicity. Overall, these results are consistent with the hypothesis that FLT3 ITD mutant AML with high allele burden or relapsed samples are more addicted to FLT3 ITD. To further examine this topic, we studied AML cell lines that express only wild type FLT3 (THP1), both FLT3-ITD and WT FLT3 (MOLM14) or FLT3-ITD but not WT-FLT3 (Mv4;11, TF1-ITD). Growth of all three cell lines expressing FLT3-ITD but not THP1 cells was inhibited by crenolanib. Crenolanib inhibited tyrosine phosphorylation and activation of downstream signaling pathways in all three FLT3-ITD+ cell lines. Importantly, crenolanib was active at 10 nM in TF1-ITD cells but higher concentrations were required to inhibit signaling in Mv4;11 cells. This suggests that allele ratio alone does not determine sensitivity to FLT3 inhibitors. Interestingly, FLT3 ligand (FL) impairs inhibition of FLT3 by kinase inhibitor and, again, mutant cell lines are similarly responsive to FL in the presence of kinase inhibitor. In summary, these data demonstrate that AML cells have variable dependence on mutant FLT3 for survival. Samples with high allele ratio and relapsed samples behave in a manner consistent with oncogene addiction and are likely to show cytotoxicity to FLT3 inhibition. However, the basis for oncogene addiction is unclear and does not depend on activation of the canonical signal transduction pathways known to be downstream of FLT3. Interestingly, a recent unbiased screen of phosphorylated proteins in FLT3-ITD+ AML to predict clinical response to AC220 did not identify tyrosine phosphorylation of canonical FLT3 targets, but correlated clinical response with serine phosphorylation on EEPD1-S160, BCL11A-S630, and RANBP3-S333 (Schaab C, et al. Leukemia 2014). Analysis of the effects of FLT3 inhibitors upon these proteins in FLT3 mutant primary samples and cell lines is ongoing. Disclosures Perl: Arog pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy.

scholarly journals Autophagy Activation Induces Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia with FLT3-ITD Mutation

2021 ◽  
Author(s):  
Dan Xu ◽  
Zhao Yin ◽  
Ying Yang ◽  
Yishan Chen ◽  
Changfen Huang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Leukemic Cells ◽  
Sorafenib Treatment ◽  
Downstream Signaling ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Inhibitor Resistance ◽  
Acute Myeloid

Abstract Background: Autophagy plays a critical role in drug resistance in acute myeloid leukemia (AML), including the subtype with FLT3-ITD mutation. Yet how autophagy is activated and mediates resistance to FLT3 inhibitors in FLT3-ITD-positive AML remains unsure. Methods: We detected the alteration of autophagy in FLT3-ITD-positive leukemic cells after versus before acquired resistance to FLT3 inhibitors; tested the stimulative effect of acquired D835Y mutation and bone marrow micro-environment (BME) on autophagy; explored the mechanism of autophagy mediating FLT3 inhibitor resistance. Results: Sorafenib-resistant cells markedly overexpressed autophagy in comparison with sorafenib-sensitive cells or the cells before sorafenib treatment. Both acquired D835Y mutation and BME activated cytoprotective autophagy to induce FLT3 inhibitor resistance. Autophagy activation decreased the suppression efficacy of FLT3 inhibitors on FLT3 downstream signaling and then weakened their anti-leukemia effect. Inhibition of autophagy with CQ significantly enhanced the suppressive effect of FLT3 inhibitor on FLT3 downstream signaling, in the end overcame FLT3 inhibitor resistance. Conclusions: Autophagy might be stimulated by acquired mutation or BME, and bypass activate FLT3 downstream signaling to mediate FLT3 inhibitor resistance in FLT3-ITD-positive AML. Targeting autophagy could be a promising strategy to overcome resistance.

The Multi-Kinase Inhibitor TG02 targets CD34+CD38-CD123+ AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1823-1823
Author(s):  
Monica Pallis ◽  
Francis Burrows ◽  
Nigel H. Russell
Keyword(s):  
Kinase Inhibitor ◽  
In Vitro Toxicity ◽  
The Novel ◽  
In Vivo Models ◽  
Erk Activation ◽  
Akt Phosphorylation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Abstract 1823 In clinical trials, FLT3 inhibitors are reported to kill circulating AML blasts, but the bone marrow is protected. We have previously reported that niche-like conditions (fibronectin and a cytokine cocktail) significantly reduced the in vitro toxicity of the FLT3 inhibitor AG1296 to AML cells. Moreover, the toxicity of AG1296 to the chemoresistant leukaemic CD34+CD38-CD123+ subset was completely abolished under niche-like conditions. The novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional CDKs and JAK2 as well as FLT3. TG02 has efficacy in in vivo models and induces apoptosis in primary AML cells. We have now evaluated its in vitro toxicity under niche-like conditions in bulk AML cells and in the CD34+CD38-CD123+ subset. In a cohort of six FLT3-ITD and five FLT3-wildtype samples, 100nM TG02 induced decreases of 30% in bulk cells and 32% in CD34+CD38-CD123+ cells, whereas AG1296 (5μM) induced a median 21% decrease in bulk cells under niche-like conditions but a 0% decrease in CD34+CD38-CD123+ cells. Lestaurtinib, sorafenib and sunitinib were used as comparators (all at 100 nM) and induced, respectively, 13%, 4% and 13% decrease in bulk cells and 10%, 0% and 8% decrease in CD34+CD38-CD123+ cells. FLT3 wildtype as well as ITD samples were targeted. In order to establish the molecular pathways involved in niche-mediated chemoresistance and its reversal, we treated primary AML samples with TG02 or AG1296 for 3 hours in the presence and absence of niche proteins; we measured activating phosphorylations of STAT3 (tyr705), STAT5 (tyr694), ERK1/2 (thr202/tyr404) and AKT(ser473). Basal levels of activating phosphorylations were generally higher in the bulk cells than the CD34+CD38-CD123+ cells, possibly reflecting the increased quiescence of the latter subset. STAT3, STAT5 and ERK1/2 phosphorylation were reduced by TG02 to a slightly greater degree than by AG1296 in bulk cells. However, in CD34+CD38-CD123+ cells this contrast was enhanced, such that AG1296 was ineffective, whereas TG02 was at least as effective as in bulk cells. Niche-like conditions induced an increase in phosphorylation of STAT5, but not of the other proteins tested. TG02 reduced this to basal levels in both bulk cells and CD34+CD38-CD123+ cells. AG1296 partially blocked niche-induced STAT5 phosphorylation in bulk cells, but not in CD34+CD38-CD123+ cells. It had no effect on ERK signalling. AKT phosphorylation was not informative. In conclusion, TGO2 is more cytotoxic than comparatively selective FLT3 inhibitors towards CD34+CD38-CD123+ AML cells as well as bulk cells under niche conditions and the toxicity is associated with downregulation of STAT3, STAT5 and ERK activation. Disclosures: Pallis: Tragara Pharmaceuticals: Research Funding. Burrows:Tragara Pharmaceuticals: Employment.

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