FMS-like Tyrosine Kinase 3 Tyrosine Kinase Inhibitors Research Articles

Abstract 806: KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation

Abstract More than 30% of patients with acute myeloid leukemia (AML) harbor a mutation with internal tandem duplication (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain of FMS-like tyrosine kinase-3 (FLT3), which results in aberrant activation FLT3 by auto-phosphorylation, and confers poor prognosis. To develop novel FLT3 inhibitors, ten novel oxazole amine derivatives were designed and synthesized based on FLT3 protein structure. Among the synthesized small molecule compounds, KI2011 exhibited selectively inhibitory activities of the class III receptor tyrosine kinases, especially FLT3, wile-type, FLT3-D835Y mutant and FLT3-internal tandem duplication (ITD) mutant isoforms with IC50 values of 25, 9, and 1.2 nM, respectively, determined by an in vitro kinase activity assay. The compound decreased FLT3 phosphorylation and its downstream signaling in MV4-11 cells harboring FLT3-ITD mutation. Treatment of KI2011 selectively inhibited cell proliferation and induced apoptosis of MV411 cells in a concentration-dependent manner, but not in K562 (FLT3 negative) cells. In addition, we found that KI2011 enhanced radiosensitivity of MV4-11 cells. The results suggest that KI2011 is a novel potent FLT3 inhibitor that may be further developed in the preclinical studies as a therapeutic agent in AML treatments. Citation Format: Hwani Ryu, Jie-Young Song, Jiyeon Ahn. KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 806.

The Future of Targeting FLT3 Activation in AML.

Internal tandem duplications (ITD) and tyrosine-kinase domain (TKD) mutations of the FMS-like tyrosine-kinase 3 (FLT3) can be found in up to one third of patients with acute myeloid leukemia (AML) and confer a poor prognosis. First discovered 20years ago, these mutations were identified as viable therapeutic targets, and FLT3 tyrosine-kinase inhibitors (TKIs) have been in development for the last decade with steadily increasing potency. However, FLT3-mutated AML often acquires resistance to the growing armamentarium of FLT3 inhibitors through a variety of mechanisms. In this review, we discuss the distinct clinical phenotype of FLT3-mutated AML, historically and currently available therapeutics, mechanisms of resistance, ongoing trials, and future outlook at treatment strategies.

FLT3 activating mutations display differential sensitivity to multiple tyrosine kinase inhibitors.

Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that normally functions in hematopoietic cell survival, proliferation and differentiation. Constitutively activating mutations of FLT3 map predominately to the juxtamembrane domain (internal tandem duplications; ITD) or the activation loop (AL) of the kinase domain and are detected in about 1/3 of de novo acute myeloid leukemia (AML) patients. Small molecule tyrosine kinase inhibitors (TKI) effectively target FLT3/ITD mutations, but some activating mutations, particularly those on the AL, are relatively resistant to many FLT3 TKI. We reproduced many of the AL or other non-ITD activating mutations and tested 13 FLT3 TKI for their activity against these and wild-type FLT3. All 13 TKI tested inhibited BaF3/ITD cell proliferation in a concentration-dependent manner as reported, but most TKI exhibited a wide range of differential activity against AL and other point mutants. Western blotting results examining inhibition of FLT3 autophosphorylation and signaling pathways indicate that many AL mutations reduce TKI binding. Most FLT3 TKI effectively target wild-type FLT3 signaling. As a demonstration of this differential activity, treatment of BaF3 D835Y cells transplanted in BALB/c mice with sorafenib showed no effect in vivo against this mutant whereas lestaurtinib proved effective at reducing disease burden. Thus, while FLT3 TKI have been selected based on their ability to inhibit FLT3/ITD, the selection of appropriate TKI for AML patients with FLT3 AL and other activating point mutations requires personalized consideration.

Signaling Adaptation to TKI Treatment Reactivates ERK Signaling in FLT3/ITD Leukemia

FMS-like tyrosine kinase-3 (FLT3) is one of the most commonly mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (FLT3/ITD) is an established driver mutation in AML and results in constitutive activation of the receptor, activating downstream signaling pathways including STAT5, PI3K/AKT, and RAF/MEK/ERK. The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3/ITD AML has been explored as a promising strategy for over a decade, but clinical responses have remained limited.In some cancers driven by mutated oncogenes, small molecule inhibition of the target is known to result in reactivation of downstream signaling pathways. This response has been observed in the context of PI3K/AKT/mTOR and BRAF inhibition, but it remains unclear whether this phenomenon extends to cancers driven by receptor tyrosine kinase (RTK) activation, such as FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response.To test this, we treated FLT3/ITD AML cell lines (Molm14 and MV4;11) with FLT3 TKIs for up to 48 hours. We observed a rebound in ERK phosphorylation beginning as early as 6 hours and continuing for the duration of treatment, while no such rebound was observed in the phosphorylation of downstream targets AKT or STAT5. When these cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed, and the anti-leukemia effects were more pronounced compared to either drug alone, both in vitro and in vivo. In vitro, the addition of low dose MEK inhibitor to FLT3 TKI treatment synergistically increased cell death and decreased cell proliferation. In a xenograft transplant model of leukemia, the addition of low-dose MEK inhibitor to FLT3 TKI treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts (p < 0.05).We next sought to determine whether rebound in ERK signaling following FLT3 inhibition was likely to occur in patients as well. We first examined whether primary leukemic cells from patients with FLT3/ITD AML also exhibit the same phenomenon observed in FLT3/ITD cell lines. After 24 hours of sorafenib treatment, the majority of samples revealed a similar rebound in ERK phosphorylation, despite sustained inhibition of phospho-STAT5. As observed with the cell lines, the addition of a MEK inhibitor to sorafenib treatment resulted in sustained pERK inhibition after 24 hours. Next, we explored whether signaling adaptation observed in culture conditions would be recapitulated by the concentrations of sorafenib present in human plasma from patients being treated with this FLT3 TKI. To replicate these conditions, we cultured Molm14 cells in plasma samples obtained from AML patients treated with sorafenib for a prolonged period. Using a variant of the plasma inhibitory activity assay, Molm14 cells were treated in either normal human plasma for one hour or patient plasma for either one or 24 hours. For all plasma samples, profound pERK rebound was observed by 24 hours after treatment, despite maximal inhibition at one hour and persistent inhibition of FLT3 and STAT5 phosphorylation over the duration.Finally, we sought to determine whether TKI-mediated rebound in ERK activity was restricted to FLT3/ITD AML or may also occur in the context of other RTK-driven cancers, such as those driven by EGFR mutation or HER2 amplification. We treated an EGFR mutant lung cancer (PC-9) and a HER2 amplified breast cancer (SKBR3) cell line with the EGFR/HER2 inhibitor lapatinib. As was seen for FLT3/ITD driven AML, pERK rebound was observed following 24 hours of TKI treatment despite persistent inhibition of the receptor and downstream target AKT. Again, the rebound could be overcome with the addition of low concentrations of MEK inhibitor.Together, these studies reveal that FLT3/ITD and other RTK driven cancer cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to upstream target inhibition. This adaptation limits the efficacy of TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML and possibly other RTK-driven cancer types. DisclosuresLevis:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria.

FLT3 TKI Are Differentially Affected By Both Plasma Binding and Stromal Survival Factors

The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML). As such it represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML, a number of which have entered clinical trials. However, to date, these trials have demonstrated limited clinical efficacy. Evidence indicates that failure to achieved adequate inhibitory activity against FLT3, at least partially as a result of plasma protein binding, as well as survival factors provided by stroma, are among the reasons for the limited efficacy of FLT3 TKI.In this study we have characterized the effects of plasma binding and stromal survival factors on TTT-3002, a novel TKI that has demonstrated potent inhibition of the internal tandem duplication (FLT3-ITD) as well as a large panel of clinically described kinase mutants (FLT3-KD), and compared it to the effects these same conditions have on other FLT3 TKI.Using plasma from pediatric/young adult patients of a variety of ages (n = 24, median 7.2 years, 4.1-36.9 years), the impact of human plasma binding on the activity of TTT-3002 against FLT3-ITD dependent proliferation. 50% human plasma results in a moderate decrease in activity of TTT-3002 (IC50 45.2 nM in human plasma, 2.4 nM in media with 10% FCS). This is a markedly smaller shift than several TKIs currently or previously in clinical trials including sorafenib, CEP-701, PKC-412, and AC-220 (50 to >500-fold increase in IC50). The data demonstrate a clear linear relationship between plasma alpha-1 acidglycoprotein (AGP), an acute-phase reactant, and fold-increase in IC50 of TTT-3002 (1.7 fold-increase per 10 mg/dL AGP). The data also demonstrate a wide variability in patient plasma AGP concentration that is dependent upon age, but also appears to be significantly impacted by position in treatment (median 117 mg/dL, 58-354 mg/dL). Plasma AGP strongly inhibits several FLT3 inhibitors including CEP-701 and PKC-412 (>100-fold increase in IC50); whereas other FLT3 inhibitors such as sorafenib and AC-220 are inhibited by human plasma in an AGP-independent manner. These data indicate the presence of several human plasma proteins whose binding leads to drug inhibition that TTT-3002 is nonetheless resistant to, in comparison to other clinically relevant FLT3 inhibitors. The identification of these proteins is ongoing.Using primary human bone marrow stromal cell co-culture with two FLT3-ITD cell lines increases the IC50 of TTT-3002 but to a lower extent than that of AC-220 (MV4-11: 1.1-1.9 fold versus 2.3-2.7 fold, MOLM-14: 2.2-2.3 fold versus 3.5-3.6 fold). The data are consistent across stromal cells cultured from several different pediatric patients (n = 5).These data suggest that TTT-3002 may be more resistant to some of the inhibitory effects that have led to disappointing results with other similar FLT3-targeted TKIs. As such, TTT-3002 represents an appealing candidate for clinical studies. DisclosuresNo relevant conflicts of interest to declare.

Pre-Clinical Activity of Novel Hypoxia-Activated FLT3 Inhibitors in FLT3-Mutated AML

Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are most frequently observed molecular abnormalities in AML, which lead to constitutive activation of the tyrosine kinase. While several mechanisms of resistance to FLT3 tyrosine kinase inhibitors (TKIs) have been identified, commonly failure is associated with lack of complete and sustained inhibition of FLT3 tyrosine kinase activity. FLT3 inhibition is particularly challenging in blasts within the hypoxic bone marrow (BM) microenvironment, which plays a crucial role in AML survival. Widespread hypoxia has been shown by us and others to be prevalent within the confines of the leukemic BM space, and to limit the efficacy of standard chemotherapy (Benito et al., PLoS One 6(8):e23108, 2011). With the goal of exploiting hypoxia as a physiological target and increasing selectivity to FLT3-mutant AML blasts, we have developed a series of hypoxia-activated prodrugs of FLT3 TKIs (HA-TKIs) based on the known clinical compounds sunitinib, AC220, MLN-518 and crenolanib. Bioreductive trigger conjugation was performed at the final step of the synthesis to provide the prodrugs as previously described (Lu et al., Tetrahedron, 69, p9130-9138, 2013). Under hypoxia, these compounds can undergo enzymatic one-electron reduction and then fragment selectively to release the cell permeable TKI, generating higher local concentrations of TKIs and limiting normal tissue exposures.Pulse radiolysis was used to determine the electron affinity (E(1)) and rate of fragmentation of each prodrug. The E(1) values ranged from -430 mV to -497 mV, while the rate of fragmentation of the prodrugs following one-electron reduction under hypoxia ranged from 28 s-1 to 400 s-1. SN37156 and SN37169 in particular demonstrated E(1) and kfrag parameters ideal for hypoxia-selective cellular metabolism (-449 mV, 85 s-1; -449 mV, 62 s-1, respectively).With the purpose of identifying the HA-TKIs with the most selective and dose-potent in vitro efficacy, we first performed an Oxic/Hypoxic screening of the hypoxia-dependent activity of HA-TKIs against FLT3-mutant AML cell lines. From the 10 compounds originally developed, SN37156 and SN37169 showed hypoxia-dependent apoptosis and growth arrest. SN37156 demonstrated the best activity against Molm13, Molm14 and MV4-11 cells under hypoxia with hypoxic cytotoxicity ratios (oxic/hypoxic IC50 ratios) of 4.9, 11 and 17.6, respectively.In our secondary screening we compared the activity of SN37156, SN37169 and their parental inhibitors on target modulation. Both HA-TKIs selectively inhibited FLT3 and ERK auto phosphorylation in MV4-11 cell line under hypoxic but not oxic conditions, with similar potency to the parental inhibitor under normal oxygen culture conditions.We next investigated the effects of both HA-TKIs on inhibition of FLT3 downstream targets, including AKT, ERK, pS6 and STAT5 using time-of-flight mass cytometry (CyTOF). The data demonstrated distinct patterns of signaling inhibition, with a dose-dependent p-ERK and p-S6 inhibition in MV4-11 and Molm-14 cells upon treatment with both HA-TKIs. In turn, both compounds at 100nM inhibited p-AKT and p-STAT5.Single dose tolerability studies were conducted in tumor-free C57BL6 mice. Prodrugs were administered by IP injection in 20% β-hydroxypropylcyclodextrin solution starting at 17.8 µmol/kg and escalating in 1.33-fold increments until lethality or severe mortality (mean body weight loss of >10%) was observed. The maximal tolerated dose for both, SN37156 and SN37169 was established at 56.1 µmol/kg. To test efficacy of HA-TKIs in vivo, we injected C57BL6 mice with genetically engineered Baf3-ITD/luc/GFP cells. Mice were randomized to treatment groups on day 8 when engraftment was documented by bioluminescent imaging (BLI), and treated with either vehicle or 56.1 µmol/kg of SN37156 or SN37169 I.P. every 3 days. Multiple doses (up to 10 so far; study is ongoing) were well tolerated. BLI demonstrated reduced leukemia burden in both treatment groups compared to control.In summary, our data indicate that HA-TKIs SN37156 and SN37169 inhibit mutant FLT3 and its selected downstream signaling targets under conditions resembling marrow hypoxia, and induce hypoxia-dependent growth arrest and apoptosis in FLT3-mutant AML cells. Planned studies in FLT3-mutant AML PDX will evaluate in vivo efficacy to eliminate primary human AML cells from hypoxic BM microenvironment. DisclosuresDaver:Kiromic: Research Funding; Ariad: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding. Konopleva:Cellectis: Research Funding; Calithera: Research Funding.

Inhibition of FLT3 in AML: a focus on sorafenib.

FMS-like tyrosine kinase 3 (FLT3) is one of the most commonly mutated genes in AML. FLT3 is mutated in ~30% of patients with AML, either by internal tandem duplications (FLT3-ITD) of the juxta-membrane domain or by a point mutation, usually involving the tyrosine kinase domain. Several FLT3 tyrosine kinase inhibitors are being evaluated in multiple studies aiming at improving outcomes. The most widely used is sorafenib, a potent multikinase inhibitor approved for hepatocellular carcinoma and renal cell carcinoma. Sorafenib monotherapy or in combination with conventional chemotherapy, has been evaluated in various settings in AML, including front-line, relapsed or refractory disease including post-allograft failures and, more recently, as post-transplant maintenance therapy. Encouraging data have emerged with several other agents like lestaurtinib, midostaurin, crenolanib, gilteritinib and quizartinib. Although transient responses to FLT3 inhibitors are often observed in case of disease relapse, the most promising approach is the use of FLT3 inhibitors either in combination with induction chemotherapy or as consolidation/maintenance therapy after allogeneic hematopoietic cell transplantation. In this review, we summarize the clinical data on sorafenib and other FLT3 inhibitors in AML.

DOCK2 interacts with FLT3 and modulates the survival of FLT3-expressing leukemia cells.

The FMS-like tyrosine kinase-3 (FLT3) gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying internal tandem duplication (ITD) mutations have a poor prognosis. Long-term inhibition of FLT3 activity in these patients has been elusive. To provide a more complete understanding of FLT3 biology, a mass spectroscopy-based screen was performed to search for FLT3-interacting proteins. The screen identified dedicator of cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rho GTPases, and its expression is limited to hematolymphoid cells. We show that DOCK2 is expressed in leukemia cell lines and primary AML samples, and DOCK2 co-immunoprecipitates with wild-type FLT3 and FLT3/ITD. Knock-down (KD) of DOCK2 by shRNA selectively reduced cell proliferation and colony formation in leukemia cell lines with increased FLT3 activity, and greatly sensitized these cells to cytarabine treatment, alone and in combination with FLT3 tyrosine kinase inhibitors. DOCK2 KD in a FLT3/ITD-positive leukemia cell line also significantly prolonged survival in a mouse xenograft model. These findings suggest that DOCK2 is a potential therapeutic target for novel AML treatments, as this protein regulates the survival of leukemia cells with elevated FLT3 activity and sensitizes FLT3/ITD leukemic cells to conventional anti-leukemic agents.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3/ITD acute myeloid leukemia cells.

Effective treatment regimens for elderly acute myeloid leukemia (AML) patients harboring internal tandem duplication mutations in the FMS-like tyrosine kinase-3 (FLT3) gene (FLT3/ITD) are lacking and represent a significant unmet need. Recent data on the effects of FLT3 tyrosine kinase inhibitors on FLT3/ITD(+) AML showed promising clinical activity, including in elderly patients. DNA methyltransferase (DNMT) inhibitors such as decitabine (5-aza-2-deoxycytidine, DEC) and 5-azacitidine (AZA) demonstrated clinical benefit in AML, are well tolerated and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 and DNMT inhibition are associated with the induction of terminal differentiation of myeloid blasts. Consequently, there is a strong theoretical rationale for combining FLT3 and DNMT inhibition for FLT3/ITD(+) AML. We therefore sought to study the anti-leukemic effects of DEC, AZA and FLT3 inhibitors, either as single agents or in combination, on AML cell lines and primary cells derived from newly diagnosed and relapsed AML patients. Our studies indicate that combined treatment using FLT3 inhibition and hypomethylation confers synergistic anti-leukemic effects, including apoptosis, growth inhibition and differentiation. The simultaneous administration of AZA and FLT3 inhibition appears to be the most efficacious combination in this regard. These drugs may provide a novel therapeutic approach for FLT3/ITD(+) AML, in particular for older patients.

Targeted Treatment of FLT3 -Overexpressing Acute Myeloid Leukemia with MiR-150 Nanoparticles Guided By Conjugated FLT3 Ligand Peptides

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. With standard chemotherapies, only 30-50% of younger (aged <60) and 5-10% of older patients with AML survive longer than 5 years. Aberrancy of FMS-like tyrosine kinase 3 (FLT3) occurs in the majority cases of AML. Two major classes of constitutively activating mutations of FLT3, i.e. internal-tandem duplications (ITDs) and tyrosine kinase domain (TKD) point mutations are found in more than 30% of AML cases and usually predict poor prognosis. Overexpression of FLT3 has also been reported in more than 70% of AML cases with a variety of AML subtypes, e.g. MLL (Mixed Lineage Leukemia)-rearranged or FLT3 -ITD AML, and may be associated with poor survival in AML patients. Given the disappointing results with FLT3 tyrosine kinase inhibitors (TKIs) in clinical trials in the past decade, decreasing the overall abundance of FLT3 at the RNA and protein levels would be an alternative strategy to treat AMLs with FLT3 overexpression and/or FLT3 -ITD/TKD mutations.MicroRNAs (miRNA) are a class of small, non-coding RNAs that play important roles in post-transcriptional gene regulation. We recently reported that miR-150 functions as a pivotal tumor-suppressor gatekeeper in MLL-rearranged and other subtypes of AML, through targeting FLT3 and MYB directly, and the MYC/LIN28/HOXA9/MEIS1 pathway indirectly. Our data showed that MLL-fusion proteins up-regulate FLT3 level through inhibiting the maturation of miR-150. Therefore, our findings strongly suggest a significant clinical potential of restoration of miR-150 expression/function in treating FLT3 -overexpressing AML.In the present study, we first analyzed FLT3 expression patterns and prognostic impact in a large cohort of AML patients (n=562). We found that FLT3 is aberrantly highly expressed in FAB M1/M2/M5 AML or AML with t(11q23)/MLL -rearrangements, FLT3 -ITD or NPM1 mutations, and that increased expression of FLT3 is an independent predictor of poor prognosis in patients with FLT3 -overexpressing AML.To treat FLT3 -overexpressing AML, we developed a novel targeted nanoparticle system consisting of FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) dendriplexes encapsulating miR-150 oligos (see Figure 1A). In FLT3 -overexpressing cell lines, the uptake ratios of the G7-FLT3L dendrimers were much higher (50.3~97.1%) than the G7-histone 2B (H2B) control nanoparticles (4.3~33.2%). And the uptake only took minutes. By integrating the miR-150 oligo with G7-FLT3L dendrimers, we constructed the G7-FLT3L-miR-150 dendriplexes, which significantly reduced the viability and increased the apoptosis of MONOMAC-6 cells carrying t(9;11) in a dose-dependent manner. To increase the stability of miR-150 oligos, we incorporated a 2'-o -methyl (2'-O Me) modification into the miRNA oligos. Indeed, the G7-FLT3L nanoparticles carrying 2'-O Me modified miR-150 exhibited a more sustained inhibition on cell growth.In order to further investigate the in vivo therapeutic effects of the miR-150 nanoparticles, we used a MLL -rearranged leukemia model. We transplanted wild-type recipient mice with primary mouse leukemic cells bearing the MLL-AF9 fusion. After the onset of leukemia, the mice were treated with G7-Flt3L or G7-NH2 control nanoparticles complexed with 2'-O Me-modified miR-150 oligos. In these treated animals, G7-Flt3L-miR-150 nanoparticles tended to be enriched in the bone marrow. The G7-Flt3L-miR-150 nanoparticles showed the best therapeutic effect (with median survival of 86 days), as compared with the control group (Ctrl; PBS treated; with median survival of 54 days) or the G7-NH2-miR-150 treated group (with median survival of 63 days). Nanoparticles carrying miR-150 mutant oligos showed no anti-leukemia effect at all. Notably, the G7-Flt3L-miR-150 treatment almost completely blocked MLL-AF9 -induced leukemia in 20% of the mice (Fig. 1B). Furthermore, the G7-Flt3L-miR-150 nanoparticles showed a synergistic effect with JQ1, a small-molecule inhibitor of the MYC pathway, in treating AML in vivo (Fig. 1C).Collectively, we have developed a novel targeted therapeutic strategy to treat FLT3-overexpressing AML, such as MLL-rearranged leukemias, which are resistant to currently available therapies, with both high specificity and efficacy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

AGS62P1, a Novel Anti-FLT3 Antibody Drug Conjugate, Employing Site Specific Conjugation, Demonstrates Preclinical Anti-Tumor Efficacy in AML Tumor and Patient Derived Xenografts

FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status.Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo.AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1.Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. DisclosuresJin:Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.

Novel Small Molecule FLT3 Inhibitors for the Treatment of FLT3-ITD AML

FMS-like Tyrosine Kinase 3 (FLT3) is a receptor tyrosine kinase that promotes growth and survival of hematopoietic stem and progenitor cells. Mutations in the gene encoding FLT3 are found in 20-30% of acute myeloid leukemias (AML), with approximately 25% of all AMLs containing an internal tandem duplication (ITD) in the juxtamembranal region. FLT3-ITD AML is associated with poor prognosis, with a 5 year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. As such, FLT3 inhibitors have been developed to treat FLT-ITD AML. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming. A Phase II study of quizartinib (AC220) showed few complete remissions in AML patients treated with AC220 as a single agent. Additionally, patients who relapse after FLT3 inhibitor treatment can develop FLT3-inhibitor resistant clones, many of which acquire a D835Y substitution. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse for FLT3-ITD AML patients.Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. After initial biochemical and functional analysis, NCGC-2327 emerged as our lead compound, and further optimized with improved solubility, stability, and permeability properties suitable for in vivo utility (NCGC-1481). Both compounds have excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5.08 x 10-10 M). AC220, NCGC-2327, and NCGC-1481 treatment of primary human FLT3-ITD-mutant AML cells show EC50 at subnanomolar concentrations (0.5 nM, 0.4 nM, and 0.1 nM respectively) as determined by CellTiter Glo proliferation assays. To ensure that our compounds were selectively effective against FLT3-ITD-mutant AML, primary human NRas-mutant AML cells treated with the inhibitors revealed an EC50 outside of the tested range (>30 µM). To assess the ability of the compounds to induce apoptosis, we treated FLT3-ITD-mutant AML cells with AC220, NCGC-2327, NCGC-1481, or Crenolanib (a FLT3 inhibitor that can inhibit FLT3-ITD-D835Y) for 72 hours. NCGC-148-treated cells showed the greatest levels of apoptosis (AnnexinV+) as compared to Crenolanib, AC220, and NCGC-2327 (P< 0.01). Consistent with the viability assays, NCGC-1481 treatment showed the greatest inhibition of leukemic progenitor function in methylcellulose (Vehicle: 189 ± 39, Crenolanib: 151 ± 32, AC220: 36 ± 3, NCGC-2327: 22 ± 3, and NCGC-1481: 3 ± 2) (P<0.01).Relapse and resistance is a primary concern for patients treated with AC220, therefore we investigated leukemic subclonal resistance in vitro after treatment with Crenolanib, AC220, NCGC-2327, or NCGC-1481. FLT3-ITD-mutant AML cells were treated for 72 hours, washed and then allowed to recover in the absence of the compounds for one week. Subclonal recovery was assessed by measuring cell viability (AnnexinV+) and leukemic progenitor function (methylcellulose) for up to 1 week post treatment. NCGC-2327 and NCGC-1481 delayed, and in some instances prevented, subclonal recovery as compared to AC220 or Crenolanib treatment.NCGC-2327 and NCGC-1481 show comparable potency to current FLT3 inhibitors (i.e., AC220 and Crenolanib) in regards to inhibition of FLT3 signaling, proliferation, and induction of apoptosis in FLT3-ITD-mutant AML. However, NCGC-2327 and NCGC-1481 are exquisitely effective at preventing subclonal recovery of FLT3-ITD-mutant AML as compared to both AC220 and Crenolanib. Taken together, these findings suggest our novel FLT3 inhibitors show promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Full Doses of Crenolanib, a Type I FLT3 Inhibitor, Can be Safely Administered in AML Patients Post Allogeneic Stem Cell Transplant

▪Introduction:Crenolanib besylate is a selective and potent type I tyrosine kinase inhibitor (TKI) of wild-type and mutant FMS-like tyrosine kinase 3 (FLT3). Crenolanib is being studied in patients with relapsed/refractory FLT3+ AML with FLT3-ITD and/or FLT3-D835 mutations. A total of 65 patients with relapsed/refractory AML have been treated with crenolanib. This retrospective analysis was done to assess whether full doses of crenolanib can be safely administered in the post allogeneic transplant setting (HSCT).Methods:Of the 65 patients treated with crenolanib, 16 patients (9 females and 7 males) had undergone HSCT prior to initiation of crenolanib. The median age was 51 years (range: 24-72). 15 patients had received salvage chemotherapies (median of 3 systemic therapies range: 1-5) following their recurrence post-transplant and prior to initiation of crenolanib. 11/16 patients also had prior exposure to FLT3 TKIs, including sorafenib (9 patients), ASP2215 (2 patients) and quizartinib (3 patients). The median time between allogeneic stem cell transplantation and initiation of crenolanib was 9 months (range, 1 to 69 months). One patient began maintenance crenolanib one month following transplant.Crenolanib was initially given at a dose of 100 mg TID (7 patients) and subsequently escalated to a higher dose of 200 mg/m2 (9 patients). The highest total dose of crenolanib was 400 mg daily (2 patients). Patients were monitored for serious adverse events while on crenolanib, specifically for elevated liver transaminases and GI toxicities. Regular assessments of chemistry and hematologic laboratory values were performed to ensure safety of crenolanib in this patient population.Results:Crenolanib was well tolerated in the post-transplant setting and none of the 7 patients treated at 300 mg daily dose of crenolanib required dose reduction. Of the 9 patients treated at the higher dose of 200 mg/m2, 4 patients (total daily doses of 400, 360, 340 and 320 mg) did require dose reductions; no patients required drug discontinuation due to toxicities attributed to crenolanib. The pharmacokinetic profile in this patient population was consistent with that seen in all patients treated with crenolanib. For those patients who received crenolanib at 100mg TID, the median Cmax was 149 nM (range: 105-624 nM). Those patients who received crenolanib at 200mg/m2 had a median Cmax of 391 nM (range: 328-1497 nM).Grade 1 and 2 nausea and vomiting were seen but were well controlled with standard anti-emetics, requiring no dose reductions or discontinuation. Two patients experienced grade 1 ALT and AST elevation and one patient experienced grade 1 ALT, AST and bilirubin elevation; no patient required dose interruption or reductions. The only grade 3 elevation of AST was attributed to GVHD and improved with treatment of GVHD and did not require crenolanib discontinuation. One patient developed grade 3 hyperbilirubinemia on day 53 of crenolanib. This patient had achieved a CRi on crenolanib and liver biopsy showed development of acute graft versus host disease. Following treatment of GVHD, the patient was able to restart crenolanib at 280 mg daily.One patient received crenolanib maintenance in the immediate post-transplant setting. This patient was able to stay on crenolanib at a dose of 280 mg a day for 5 months with full recovery of neutrophils and platelet counts, and became transfusion independent.Conclusion:Post-transplant maintenance with a FLT3 inhibitor can potentially be beneficial in reducing post-transplant recurrence and improving overall outcomes. This retrospective study shows that crenolanib appears to be well-tolerated in patients with leukemia who have undergone a prior allogeneic transplant at the full therapeutic dose of 100 mg TID (300 mg daily). A formal study of crenolanib maintenance post-allogeneic stem cell transplantation has been initiated (NCT 02400255). DisclosuresChen:Arog Pharmaceuticals: Employment. Ramachandran:AROG Pharmaceuticals: Employment. Cortes:Teva: Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Abstract C78: ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for approximately 20% of childhood leukemias. Although frontline treatment of AML with cytotoxic chemotherapy is capable of achieving high remission rates, 75-80% of patients will either not respond to or will relapse after initial therapy, and most patients will die of their disease. More effective and better-tolerated therapies for AML are required. Activating mutations in fms-like tyrosine kinase 3 (FLT3) are the most frequently observed genetic defect in AML and drive leukemic cell growth and survival. FLT3 tyrosine kinase inhibitors have shown impressive anti-leukemic activity in clinical trials, however, sustained remissions using these inhibitors as monotherapy have not been achieved. In order to identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a large-scale RNA interference-based screen. The screen identified several genes involved in metabolic regulation, including ataxia telangiectasia mutated (ATM), as being synthetic lethal with FLT3 inhibition in FLT3 mutated AML. Genetic or pharmacological inactivation of ATM or its downstream effector glucose-6-phosphate dehydrogenase (G6PD) sensitized AML cells to FLT3 inhibition through enhancing apoptosis. Whole metabolome profiling revealed that FLT3 inhibition causes severe and widespread metabolic deficiencies, including depletion of the antioxidant factor glutathione. Inactivation of either ATM or G6PD exacerbated glutathione depletion upon FLT3 inhibition. Subsequent analyses revealed that FLT3 inhibition elicits severe mitochondrial oxidative stress that is causative in apoptosis and accentuated by ATM or G6PD inhibition. The use of a drug that promotes the production of mitochondrial reactive oxygen species (ROS) in combination with a FLT3 inhibitor augmented elimination of AML cells both in vitro and in vivo. Our data support the hypothesis that FLT3 mutated AML cells are highly dependent on FLT3 activity to maintain glutathione levels and sufficient mitochondrial antioxidant capacity to sustain cell survival. Moreover, these data support the novel strategy of employing mitochondrial ROS inducing agents as adjuvant to FLT3 inhibitor therapy to more effectively treat FLT3 mutated AML. Citation Format: Mark A. Gregory, Angelo D'Alessandro, Francesca Alvarez-Calderon, Jihye Kim, Travis Nemkov, Aik Choon Tan, Kirk C. Hansen, James DeGregori. ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C78.

Genetic and Pharmacologic Notch4 Inhibition Synergizes with FLT3 Tyrosine Kinase Inhibition in Vitro to More Effectively Eliminate FLT3/ITD-Positive Leukemia Cells

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. Mutated in approximately 1/3 of patients, FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML). The presence of FLT3 internal tandem duplication (ITD) mutations, which renders FLT3 constitutively active, confers a particular poor prognosis. While several FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling, the clinical success of these drugs is limited because they fail to achieve frequent complete durable responses, despite achieving high levels of FLT3 inhibition. These limited clinical responses suggest that monotherapy is unlikely to be curative. Current clinical trials of FLT3 inhibitors are administered in combination with chemotherapy to achieve success in inducing complete remissions. While there is hope that FLT3 inhibition in combination with chemotherapy will increase cure rates, it would be optimal to achieve cures without chemotherapy altogether and its inherent toxicities. Towards this goal it is necessary to uncover critical genes/pathways that collaborate with FLT3 mutations to transform cells. In recent years, the use of whole genome sequencing has greatly expanded the list of genes mutated in AML. However, many of these mutations do not represent a practical therapeutic target because they are present in a low frequency in the patient population, making molecular targeted therapy for each of these improbable to achieve. We hypothesize that these mutated genes likely funnel into a more limited number of signaling pathways, with some pathways being more important than others in contributing to FLT3/ITD AML. Identifying FLT3/ITD cooperative pathways whose inhibition might synergize with FLT3 inhibition could hold promise for greatly improving the cure rate in FLT3 mutant AML.We performed a loss of function RNAi screen in FLT3/ITD+ AML cell lines (Molm14 and MV411) to uncover genes and pathways whose inhibition combined with FLT3/ITD inhibition to more effectively kill FLT3 mutant AML cells. Notch4 signaling was identified as one such potential target. We found Notch4 to be overexpressed in FLT3/ITD+ AML cells lines as well as in the Lin-Sca+Kit+ fraction of bone marrow isolated from FLT3/ITD+ mice. Stable, tetracycline-inducible Notch4 knockdown cell lines were established in Molm14 and MV411 cell lines. While Notch4 knockdown alone slightly decreased cell growth, Notch4 knockdown in combination with FLT3 TKI demonstrated a significant decrease in proliferation compared to FLT3 TKI treated cells expressing a scrambled shRNA control. Additionally, colony forming unit assays revealed that Notch4 knockdown cells treated with FLT3 TKIs exhibited a decrease in clonogenicity compared to FLT3 TKI treated control cells. Pharmacologic inhibition of Notch4 by treatment with gamma secreatse inhibition (GSI) was also tested. Molm14, MV411, and THP-1 (FLT3 wild type AML) cells were treated with FLT3 TKIs (sorafenib, CEP-701, or AC220) alone, GSI alone, FLT3 TKI plus GSI, or vehicle control and assessed for anti-proliferative and apoptotic effects by MTT, and annexin V/7-AAD staining, respectively. In both assays, the combination of FLT3 TKI and GSI exhibited synergy with combination index (CI) values <1. Furthermore, Western blot analysis showed that while treatment of Molm14 and MV411 cells with GSI had little effect on FLT3 signaling (pSTAT5, pAKT, pMAPK activation), the combination of GSI plus FLT3 TKI inhibited FLT3 signaling significantly more than treatment with FLT3 TKI alone. The results of these experiments identify Notch4 signaling as a potential pathway whose inhibition can synergize with FLT3 inhibition to more effectively kill FLT3/ITD+ AML cells. Combining FLT3 inhibition with Notch4 inhibition may have potential for improving the cure rate of patients with FLT3 mutant AML. DisclosuresNo relevant conflicts of interest to declare.

FLT3 kinase inhibitor TTT-3002 overcomes both activating and drug resistance mutations in FLT3 in acute myeloid leukemia.

There have been a number of clinical trials testing the efficacy of FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors (TKI) in patients with acute myeloid leukemia (AML) harboring a constitutively activating mutation in FLT3. However, there has been limited efficacy, most often because of inadequate achievement of FLT3 inhibition through a variety of mechanisms. In a previous study, TTT-3002 was identified as a novel FLT3 inhibitor with the most potent activity to date against FLT3 internal tandem duplication (FLT3/ITD) mutations. Here, the activity of TTT-3002 is demonstrated against a broad spectrum of FLT3-activating point mutations, including the most frequently occurring D835 mutations. The compound is also active against a number of point mutations selected for in FLT3/ITD alleles that confer resistance to other TKIs, including the F691L gatekeeper mutation. TTT-3002 maintains activity against patients with relapsed AML samples that are resistant to sorafenib and AC220. Studies utilizing human plasma samples from healthy donors and patients with AML indicate that TTT-3002 is only moderately protein bound compared with several other TKIs currently in clinical trials. Tumor burden of mice in a FLT3 TKI-resistant transplant model is significantly improved by oral dosing of TTT-3002. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKIs in the treatment of FLT3-mutant AML. Cancer Res; 74(18); 5206-17. ©2014 AACR.

TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo

AbstractMore than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML.

Pharmacological Inhibition Of The SIRT1 Deacetylase With The Small Molecule Inhibitor Tenovin-6 Enhances Ablation Of FLT3-ITD+ LSC In Combination With TKI Treatment

Acute myeloid leukemia (AML) is propagated by small populations of leukemia stem cells (LSC). Internal tandem duplication (ITD) in the FMS-like tyrosine kinase-3 (FLT3) is the most frequent mutation seen in AML patients, and is associated with poor prognosis. However, FLT3 tyrosine kinase inhibitors (TKI) demonstrate only moderate clinical activity in FLT3-ITD AML patients. Persistent FLT3-ITD+ AML LSC may represent a source of relapse after treatment. Additional therapeutic strategies are required to target LSC and improve outcomes for FLT3-ITD+ AML patients. We have shown that the stress-related deacetylase SIRT1 is expressed at high levels in FLT3-ITD+ AML stem/progenitor cells and in cord blood CD34+ cells ectopically expressing FLT3-ITD. Inhibition of SIRT1 using RNAi or a potent small molecule inhibitor Tenovin-6 (TV-6) induces apoptosis in FLT3-ITD+ AML progenitors via p53-dependent mechanisms while sparing normal CD34+ cells suggesting a potential role for SIRT1 in FLT3-ITD+ AML LSC maintenance (Blood 2012,120: A1368). We now show that treatment of FLT3-ITD+ AML CD34+ cells with the TKI AC220 (20 nM) did not significantly reduce SIRT1 levels, increase p53 acetylation, or increase p53 target gene expression (p21, Bax, Puma, DR5, Noxa), suggesting that kinase independent mechanisms maintain SIRT1 expression and activity in TKI-treated cells. The combination of TV-6 (1μM) with AC220 (20nM) significantly reduced survival of primary human FLT3-ITD+ AML CD34+ cells compared with AC220 or TV-6 alone (combination 47±8% inhibition, AC220 alone 21±7%, TV-6 alone 25±5%, p=0.03, n=7), and significantly increased inhibition of AML CD34+ cell growth compared to AC220 and TV-6 alone (TV-6 44±7% inhibition; AC220 30±6%, TV+AC220 59±8%, p=0.04, n=7). TV-6 treatment increased p53 expression and activity in AC220-treated FLT3-ITD+ AML CD34+ cells, but did not affect FLT3 phosphorylation or STAT5 activation. Interestingly, TV-6 treatment also reduced survival of FLT3-ITD+ AML CD34+ cells expressing the D835Y mutation associated with TKI resistance (AC220 5±5% inhibition, TV 33±8%, p=0.04, n=3], suggesting SIRT1 inhibition can target TKI-resistant FLT3-ITD+ AML cells. We next tested the effect of TV-6, AC220 and the combination on primary cells from one untreated cytogenetically normal and FLT3-ITD+ patient engrafted in NSG mice (2×106 cells/mouse).12 weeks after transplantation, mice were treated with vehicle, AC220 (10mg/kg, gavage), TV-6 (100mg/kg, intraperitoneal), or the combination (n=7 per group) for 4 weeks. Treatment with TV reduced human cell engrafted in NSG mice (TV-6 2.9*107±1.2*107 human CD45+ cells/2 femurs vs. Control 5.3*107±2.6*107, after 4 weeks treatment, p=0.04). Importantly, the combination of TV and AC220 significantly reduced AML cell engraftment compared with AC220 alone (AC220 2.4*107±0.9*107 human CD45+ cells. Combination 1.2*107±0.6*107, p=0.01). Residual functional LSC were assessed by transplantation into secondary recipients. BM cells from the combination treatment group demonstrated significantly reduced engraftment of leukemia cells after secondary transplantation compared to AC220 treatment alone (16 weeks after secondary transplantation, AC220 1.8*106±0.6*106 human CD45+ cells/2 femurs, Combination 0.4*106±0.1*106, p=0.02, n=6). To determine the potential of this combination for FLT3-ITD+ AML patients refractory to standard chemotherapy, AML xenografts established using cells from a chemotherapy-resistant patient (n = 36) were treated with TV-6, AC220, or the combination vs. Standard of care (SOC, Ara-C + DNR), and vehicle control. Molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies after 4 weeks of therapy revealed significant differences between combination and single arm groups (vs. TV-6, p=0.0005, n=8; vs. AC220, p=0.003, n=7). Furthermore, TV-6 alone increased survival over controls (TV-6 vs. Control, p=0.02, n=6), and the combination demonstrated significantly improved survival over AC220 (p=0.02) and SOC (p=0.006. n=6). In conclusion, our studies indicate that elevated SIRT1 expression in FLT3-ITD AML cells is maintained after FLT3 TKI treatment. Inhibition of SIRT1 with TV-6 can enhance ablation of FLT3-ITD+ AML LSC in combination with FLT3 TKI treatment. Our results support further evaluation of inhibition of SIRT1 as a therapeutic strategy in FLT3-ITD+ AML. Disclosures:Popa:KinN Therapeutics AS: Employment.

FLT3 inhibitors for acute myeloid leukemia: a review of their efficacy and mechanisms of resistance

Since the Food and Drug Administration approval of imatinib for treatment of chronic myeloid leukemia in 2001, tyrosine kinase inhibitors (TKIs) have become a mainstay in the care of many malignancies. In acute myeloid leukemia (AML), activating mutations in the FMS-like tyrosine kinase 3 (FLT3) gene result in survival and proliferation of leukemic blasts and are associated with adverse prognosis. Therefore, the FLT3 receptor is an appealing target for inhibition. Multiple small molecule TKIs are currently in development for FLT3-mutated AML, and agents are beginning to show promising efficacy. In other malignancies, the development of resistance to TKIs during the course of therapy has proven to be a challenge, and thus far, in clinical trials of FLT3 TKIs, resistance to inhibition represents a significant barrier to successful FLT3 inhibition. Understanding the mechanisms of resistance and overcoming these obstacles to target inhibition will be central to the success of these agents.