FLT3 TKI Research Articles

Metformin Potentiates Gilteritinib Sensitivity Via Targeting PLK1 Signaling: A Strategy to Improve Outcomes and Reduce Costs in Treating FLT3-Mutated Acute Myeloid Leukemia

FLT3 mutations (including FLT3-ITD and FLT3-TKD), occurring in ~30% of acute myeloid leukemia (AML) patients, signal poor prognosis due to elevated relapse risk. Albeit FLT3 tyrosine kinase inhibitors (FLT3 TKI) demonstrated efficacy in treating FLT3-mutated AML in the clinic, there are issues such as high cost, side effects, and high rate of drug resistance/relapse. Hence, an affordable, efficient treatment with lower side effects is needed. Metformin (MET), a cost-effective anti-diabetic drug with minimal side effects, has demonstrated anti-tumor effects (Dowling R et al. Cancer Res. 2007). Here we aimed to determine if combining MET with FLT3 TKI, like Gilteritinib (GLT), could enhance efficacy, reduce side effects and costs in treating FLT3-mutated AML. To this end, we first assessed the IC50 values of MET and GLT across various AML cell lines including those carrying FLT3/ITD (MOLM13, MV4-11, and MA9.3-ITD), FLT3 TKI resistant cells (MOLM13-RES) and those carrying FLT3 wild-type (FLT3/WT) (THP1, NOMO1 and Kasumi-1). MET's IC50 S were consistent (7.284 -12.76 mM) (Fig. 1A), while GLT's varied, with lower values in FLT3/ITD cells (10 nM) and higher in FLT3 TKI resistant (241 nM) and FLT3/WT cells (334-1,053 nM) (Fig. 1B). Using the Chou-Talalay Method, a Combination Index (CI) below 1 in FLT3/ITD and MOLM13-RES cells indicated synergy, whereas it showed antagonism (CI>1) in FLT3/WT cells (Fig. 1C). Both MET and GLT induced cell cycle arrest and apoptosis, with Combo1 (GLT 5 nM + MET 5 mM) matching GLT 10 nM effects; Combo2 (GLT 10 nM + MET 10 mM) amplified these effects (Fig. 1D and E). We next evaluated the potential synergistic effect of MET and GLT in treating TKI-resistance AML in vivo using NRGS mice xeno-transplanted with MOLM13-RES cells. Both bioluminescence imaging and flow cytometry assays showed each treatment alone reduced leukemia burden, and their combination had a synergistic effect, reducing AML burden dramatically (Fig. 1F). While MET or GLT alone doubled overall survival compared to vehicle treatment (56-62 days vs. 26 days), ~90% of the mice treated with the Combo survived over 120 days (Fig. 1G) and their leukemia burden was still minimal, highlighting the synergistic effect and the promising therapeutic potential of this combination in treating FLT3-mutated AML (even with TKI-resistance). To understand the underlying molecular mechanism(s), we performed RNA-seq with vehicle- or drug-treated MOLM13-RES cells, followed by pathway analysis and validation/mechanistic studies, we identified Polo-like kinase 1 (PLK1) as a key target of MET and GLT (Fig. 1H). PLK1's expression is notably suppressed by both treatments (Fig. 1I). Mimicking MET treatment, PLK1 knockdown enhanced GLT sensitivity, reducing IC50 from 259.2 nM to 47-60 nM (Fig. 1J). Conversely, PLK1 overexpression increased GLT's IC50 7-fold (13.07 nM to 95.94 nM) (Fig. 1K), largely reversing MET's effect (data not shown). PLK1, a crucial serine/threonine-protein kinase, influences multiple signaling pathways such as MAPK and mTOR pathways. Upon treating MOLM13 or MOLM13-RES cells with MET and GLT alone or combined, the PLK1 levels and phosphorylation levels of FLT3, STAT5, ERK, and mTOR were decreased, with the most significant decrease occurred upon the combined treatment (Fig. 1L). Depletion and overexpression of PLK1 decreased and increased these phosphorylation levels, respectively (data not shown). Together, our findings suggest that the synergistic therapeutic effect of MET and GLT in treating FLT3-mutated AML is likely through synergistically targeting PLK1 and its downstream pathways. Meanwhile, we executed a retrospective clinical study of FLT3/ITD AML patients who were also diagnosed with diabetes. Metformin users showed a notably longer median OS (13.2 months) than non-MET users (4.5 months, P=0.0197) (Fig. 1M). Despite FLT3/ITD status, FLT3 TKIs were seldom used in China due to high cost. Additionally, PLK1 level was dramatically higher in FLT3/ITD AML cases than in both healthy and FLT3/WT cases (P<0.001) (Fig. 1N), implying PLK1's role in FLT3/ITD's poor prognosis. In summary, our findings suggest a potential synergistic approach in treating FLT3-mutated AML using MET and GLT. This approach could reduce side effects and costs (crucial for developing countries) and improve outcomes. Larger clinical trials are needed to confirm these promising initial findings.

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

Abstract 5665: HX301 (ON1232580) a novel kinase inhibitor with potent activity against CSF1R and FLT3, shows strong anti-AML activity in defined preclinical models

Abstract Acute myeloid leukemia (AML) is an aggressive leukemia of myeloid lineage with different subtypes of varying treatments/outcomes and a global annual mortality ~150,000. The first-line treatment of AML is usually induction chemotherapy, followed by further chemo-/radiation therapies or stem cell transplant. Targeted therapy tailored for specific driver mutations could be alternative treatment options, e.g., new inhibitors targeting IDH mutations and FLT3-ITR CSF1R is a receptor tyrosine kinase (RTK) responsible for the growth, survival and polarization of certain myeloid lineages of cells including macrophages. It is also frequently expressed in certain AML patient populations, thus implicated in the pathogenesis, or a new possible drug target of AML. To test this hypothesis, a novel kinase inhibitor, HX301 with strong anti-CSF1R activity (IC50 of ~0.7nM) as well as anti-FLT3 (IC50 of ~7nM), were assessed for anti-AML activity. First, an in vitro proliferation assay was performed using primary macrophages and a panel of AML cell lines, confirming that HX-301 has high potency in primary macrophage culture (IC50 of ~70nM) and also among cultures of a subsets of AML lines (e.g. IC50 < 1µM), such as MV4-11 (medium CSF1R expressing and FLT3-ITD), EOL1, MOL13, etc. Next, HX301 was pharmacologically modeled using four preclinical models to test anti-leukemia activity in vivo, including subcutaneous xenograft of MV4-11 cells (CDX) and systemically engrafted PDX models AM8096 (high CSF1R expression but wild type FLT3), AM7577 (FLT3-ITD but little CSF1R expression) and AM5512 (wild-type FLT3 and little CSF1R expression). Our data demonstrated that HX301 partially inhibited MV4-11 tumor growth as measured by tumor volume, consistent with the in vitro observation, possibly due to the inhibition of CSF1R or FLT3-ITD, or both. On the other hand, HX301 completely suppressed leukemogenesis of AM8096, likely due to the inhibition of CSF1R. This observation suggested that CSF1R is likely the driving mechanism for the leukemogenesis of this model; or in another word, for being a PDX, CSF1R likely drives pathogenesis in the original patient. HX301 also suppressed AM7577 growth, likely due to the suppression of FLT3-ITD activity since we previously reported FLT3-ITD being the driver mutation in this model which responded to AC220, a FLT3 TKi. Lastly, HX301 has little activity against AM5512. All the data suggests that HX301 can potentially be explored for the treatment of AML, at least a subset of the patients with CSF1R and FLT3-ITD as leukemogenic drivers. Further preclinical/translational studies are being conducted in order to reveal predictive biomarkers, in addition to FLT3 mutation and CSF1R expression/mutations. We believe that HX301 could be a potential candidate for treating subset of AML, warranting further clinical investigation. Citation Format: Xiaoyu An, Henry Li, Linda Xue, Jinping Liu, Lingxin Xiong, Hang Ke, Cen Chen, Bing Gao, Jia Zheng, Zhengzheng Bao, Sheng Guo, Lei Zhang, Faming Zhang. HX301 (ON1232580) a novel kinase inhibitor with potent activity against CSF1R and FLT3, shows strong anti-AML activity in defined preclinical models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5665.

Autophagy Targeting and Hematological Mobilization in FLT3-ITD Acute Myeloid Leukemia Decrease Repopulating Capacity and Relapse by Inducing Apoptosis of Committed Leukemic Cells.

Simple SummaryOne of the most frequent molecular anomalies in acute myeloid leukemia (AML) is the mutation of the fms-like receptor tyrosine kinase 3 through internal tandem duplications, giving rise to a constitutive proliferative signaling. Even though clinical trials have shown that targeting this mutated kinase is of interest and well tolerated, there is still a high frequency of relapse. The emergence of AML cells upon treatment is linked to their maintenance through resistance and persistence mechanisms. Because FLT3-ITD AML cells require autophagy, we explored the consequence of autophagy inhibition by blocking the PI3-kinase class III, Vps34, when AML cells were committed. Results in vitro, ex vivo and in vivo suggest that remission with low minimal residual disease in FLT3-ITD AML offers a promising therapeutic window to target persistent leukemic cells.Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.

Efficacy and Synergy of Small Molecule Inhibitors Targeting FLT3-ITD+ Acute Myeloid Leukemia.

Simple SummaryFLT3-ITD mutations belong to the most frequent yet most detrimental genetic alterations in AML. Next-generation FLT3 inhibitors are potent therapeutics and often effective in AML patients carrying the FLT3-ITD driver kinase. However, AML cells are particularly quick in acquiring resistance to FLT3 kinase blockers. We aimed to identify novel therapeutic options for FLT3-ITD+ AML, to investigate possible emerging resistance mechanisms to FLT3 inhibitors and to explore alternative targeting strategies. We applied a kinase-focused drug screen to find alternative therapeutics. We identified ispinesib, a kinesin spindle blocker, and kinase blockers WS6, ponatinib and cabozantinib, as very efficacious agents against FLT3-ITD+ AML cells. Importantly, we identify the combination of cabozantinib and ispinesib as particularly potent against FLT3-ITD+ AML. We suggest that a combinatorial treatment with these drugs could overcome resistance mechanisms and kill FLT3-ITD+ AML blasts.Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

Phase II Trial of Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Outcomes in Genomic Subgroups

Phase II Trial of Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Outcomes in Genomic Subgroups

The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML

The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML

CXCL12 Knock-out Enhances Leukemia Stem Cell Response to Combination Chemotherapy Plus Tyrosine Kinase Inhibition in Flt3-ITD Acute Myeloid Leukemia

One of the most common mutations in adult AML is a constitutively activating internal tandem duplication in the juxtamembrane domain of the Flt3 receptor (Flt3-ITD), which portends poor prognosis due to high recurrence rates and defines a distinct subtype of disease with unique features and biology. Although several FLT3 TKIs have been developed for clinical use, responses to these drugs, especially as single agents, are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML, as well as primary Flt3-ITD TET2 mutant AML patient samples, to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. In the animal model, administration of pIpC leads to deletion of TET2 and development of AML characterized by leukocytosis, accumulation of blasts, splenomegaly, anemia, thrombocytopenia, and lethality. Limiting dilution transplantation of FACS-sorted ST-HSC, MPP and GMP populations revealed that LIC were absent from GMPs and almost exclusively limited to the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD TET2 mutant AML patients LIC capacity was restricted to primitive HSPC populations (Lin-CD34+CD38-), and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We characterized bone marrow stromal cells in Flt3-ITD AML mice by flow cytometry on collagenase digested bone fragments. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found expansion of several stromal populations in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+) and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). CXCL12 expression, however, was greater than 2-fold higher in osteoprogenitors and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing niche in supporting Flt3-ITD AML LSC. To assess the effect of CXCL12 deletion from the marrow microenvironment on AML TKI response, we transplanted murine AML cells into CXCL12flox/flox UBC-cre mice and control Cre-ve mice. We found that AML developing in Cre-ve control mice was resistant to single agent Flt3 TKI (AC220, Quizartinib) treatment, but that CXCL12 deletion modestly improved response to TKI. We next tested a combination of standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and AC220, and found that this approach resulted in more effective and selective, but only partial, reduction of leukemia cells in this model. We found that control AML mice showed an initial response to combination chemo + TKI, but developed disease recurrence by 3 weeks of treatment. In contrast, CXCL12-deleted AML mice maintained peripheral blood response for up to 3 weeks, and showed enhanced suppression of LIC-containing populations compared to control mice. We are now performing secondary transplants using BM cells harvested from these treated mice to assess long-term effects on leukemia stem cell capacity. We are also testing the effect of the combination of chemotherapy and TKI following osteoblast-specific deletion of CXCL12, using CXCL12flox/flox BGLAP-cre mice, to assess whether osteoblastic cells are the source of CXCL12 responsible for this effect. In conclusion, our results suggest that LSC in Flt3-ITD AML are found within a primitive phenotypic ST-HSC population as opposed to GMP populations as seen in some other types of AML. Furthermore, CXCL12-expressing bone marrow microenvironmental cells contribute to drug resistance in AML LSC and global knockout of CXCL12 enhances drug response in these populations. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Quizartinib with Decitabine +/- Venetoclax Is Highly Active in Patients (Pts) with FLT3-ITD Mutated (mut) Acute Myeloid Leukemia (AML): Clinical Report and Signaling Cytof Profiling from a Phase IB/II Trial

Quizartinib with Decitabine +/- Venetoclax Is Highly Active in Patients (Pts) with FLT3-ITD Mutated (mut) Acute Myeloid Leukemia (AML): Clinical Report and Signaling Cytof Profiling from a Phase IB/II Trial

Outcomes with Sequential FLT3-Inhibitor (FLT3i) Based Therapies in Patients (pts) with FLT3-Mutated Acute Myeloid Leukemia (AML) Exposed to Prior FLT3i Based Therapies

Outcomes with Sequential FLT3-Inhibitor (FLT3i) Based Therapies in Patients (pts) with FLT3-Mutated Acute Myeloid Leukemia (AML) Exposed to Prior FLT3i Based Therapies

Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms.

FLT3-ITD mutations occur in 20–30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.

Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

GADD45G ACTS AS A NOVEL TUMOR SUPPRESSOR IN AML

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China (People's Republic) Acute myeloid leukaemia (AML) is an aggressive hematologic neoplasm with distinct molecular and genetic features which is characterized by differentiation blockade and uncontrolled proliferation of malignant clones of immature myeloid cells. The growth arrest and DNA-damage induced 45 (GADD45) family protein GADD45g, has been shown to mediate DNA repair, cell cycle, senescence and apoptosis in response to various physiological and environmental stressors. Altered expression of GADD45g has been observed in multiple types of solid tumors, the function of GADD45g in hematopoietic malignancies remains almost completely unknown. Here, we found that GADD45g expression was preferentially silenced in AML, especially in AML with MLL rearrangements and FLT3-ITD mutations, and reduced expression of GADD45g correlates with poor prognosis in AML patients. Ectopic expression of GADD45g suppresses leukemic growth, induced apoptosis, senescence and differentiation of AML cells in vitro and in vivo. What's more, GADD45g caused downregulation of DNA repair proteins, resulting in inhibition of DNA double-strand break repair pathways and leading to DNA damage accumulation in AML cells. Moreover, GADD45g is epigenetically silenced in AML by both promoter DNA methylation and histone deacetylation, while the HDACi is more powerful. Exogenous expression of FLT3-ITD and MLL-AF9 repress GADD45G expression in AML, and the expression of GADD45G could be significantly induced by the small-molecule inhibitors FLT3 TKI or BRD4i target FLT3 or MLL respectively. Furthermore, the combination of HDACi and FLT3 TKI or BRD4i exerted a synergistic effect against FLT3-ITD+ or MLL-rearranged AML, respectively. These findings suggest that GADD45G may act as a tumor suppressor in AML and provide a novel and promising therapeutic target for AML.

Effect of cytarabine/anthracycline/crenolanib induction on minimal residual disease (MRD) in newly diagnosed FLT3 mutant AML.

7016 Background: Baseline characteristics such as age >60, WBC>100,000/µL and FLT3/NPM1/DNMT3A+ve are known to be associated with a poor prognosis in AML. Ivey et al. (NEJM 2016) reported that FLT3-ITD+ve patients (pts) who were MRD+ve after 2 cycles of induction chemotherapy were more likely to relapse as compared to those who became MRD-ve (92% vs 35%). Eradication of FLT3+veclones may lead to reduced relapse rates. Crenolanib is a type I FLT3 TKI, which inhibits both FLT3-ITD and TKD mutations. We here report that a single induction cycle of cytarabine/anthracycline/crenolanib leads to MRD negativity by multiparameter flow cytometry (MPF), and low rate of early relapse in pts with newly diagnosed FLT3+veAML. Methods: This abstract includes 29 consecutively treated, newly diagnosed, FLT3+ve AML pts, who achieved CR1 after one course of cytarabine/anthracycline/crenolanib. Pts received 7+3 induction with cytarabine 100 mg/m2/d for 7d and either daunorubicin (<60 y: 90 mg/m2; ≥60 y: 60 mg/m2) or idarubicin 12 mg/m2 for 3d. Crenolanib (100 mg TID) was started on day 9 until 72 h prior to next chemotherapy. Results: 29 pts (15M, 14F), median age 55y (10pts ≥60y) are included. MRD at time of count recovery was assessed by MPF in 25/29 pts. 20/25 (80%) became MRD–ve. With a median follow up of 7mth, 4/25 pts have relapsed (2/5 MRD+ve, 2/20 MRD-ve).Age ≥60 was a risk factor for MRD+ve and relapse. All 4 pts with WBC>100K as well as 5 pts with FLT3/NPM1/DNMT3A+ve AML became MRD-ve after one induction cycle and none have relapsed. Conclusions: These data suggest, in the context of an ongoing trial (NCT02283177), crenolanib in combination with standard induction is associated with a high rate of achieving an MRD negative state by MPF and a low rate of relapse in previously untreated adults with mutant FLT3. Longer follow-up and comparison of MRD data with similar pts treated with standard chemo alone will be necessary to reach more definitive conclusions. Clinical trial information: NCT02283177. [Table: see text]

What are the latest advancements in acute myeloid leukemia therapy?

What are the latest advancements in acute myeloid leukemia therapy?

Crenolanib, a Type I FLT3 TKI, Can be Safely Combined with Cytarabine and Anthracycline Induction Chemotherapy and Results in High Response Rates in Patients with Newly Diagnosed FLT3 Mutant Acute Myeloid Leukemia (AML)

Background: Crenolanib is a type I oral FLT3 TKI, which inhibits both FLT3-ITD and FLT3-TKD mutations (D835, N841, etc). Crenolanib has a half-life of 6-8 hours and does not accumulate with repeated dosing. Crenolanib does not inhibit c-kit at clinically achievable concentrations, potentially allowing for full count recovery even when combined with myelosuppressive chemotherapy. We report here the first analysis of a phase II trial evaluating the tolerability and efficacy of crenolanib combined with standard induction chemotherapy in patients (pts) with newly diagnosed FLT3 mutant AML.Methods: Pts (≥ 18yrs) (no upper age limit) with newly diagnosed AML characterized by FLT3 (ITD and/or TKD) mutations were enrolled. Pts received standard 7+3 induction with cytarabine 100 mg/m2/d for 7 days and either daunorubicin (<60yrs: 90 mg/m2; ≥60yrs: 60 mg/m2) or idarubicin 12 mg/m2 for 3 days. Crenolanib 100 mg TID was administered starting on day 9 until 72 hrs prior to next chemotherapy. Re-induction was permitted for pts with inadequate leukemia cytoreduction. Up to 4 courses of consolidation consisting of 6 doses of high dose cytarabine (HiDAC) (<60yrs: 3 g/m2; ≥60yrs: 1g/m2) was given, with crenolanib starting on day 7. Eligible pts could proceed to allogeneic stem cell transplant (SCT). Crenolanib maintenance therapy was offered for 1 year after HiDAC or -SCT. Safety and tolerability were assessed as well as anti-leukemic activity.Results:Demographics: Twenty-six pts (14 females, 12 males) with a median age of 55yrs (range 22-74yrs) have received 7+3 followed by crenolanib; nine (35%) pts were ≥ 60yrs. Five pts had initial WBC >100,000/μL (two pts had WBC >200,000). Three pts had AML following antecedent MDS or MPN.Mutational Analysis: Twenty-two pts had either FLT3-ITD (n=19) or FLT3-D835 (n=3) mutations. Genomic sequencing revealed multiple concurrent FLT3 activating mutations in four pts. One pt had a dual-activating kinase domain mutation in trans (D835Y+N841T). Three other pts had FLT3-ITD together with N841 (n=2), I836del (n=2), and V592/593 (n=2). Fifteen (60%) pts had FLT3 and NPM1 mutations, and 11 (52%) had FLT3 and DNMT3A mutations. Five pts (24%) had AML with three mutations (FLT3 + NPM1+ DNMT3A). WIT (WT1, IDH1/2, TET2) mutations were present in 11 (52%) pts. Three pts had trisomy 8; one pt each having monosomy 7, trisomy 4, and t(3;18)del(6q)der(3).Treatment Outcome: Eighteen (69%) pts received induction with cytarabine+daunorubicin (10 pts at 90 mg/m2, 8 pts at 60 mg/m2) and eight pts (31%) received cytarabine+idarubicin. To date, crenolanib has been well-tolerated in combination with chemotherapy. Six pts required crenolanib dose reductions for periorbital edema (n=2), delayed count recovery (n=1), LFT elevation (n=1), nausea (n=1) and rash (n=1). Out of the twenty-five pts evaluable for response, twenty-two (88%) achieved complete remission (CR) with full count recovery after one cycle of induction. Overall CR/CRi rate was 96% (Table 1).Sixteen pts (10 pts< 60yrs; 6 pts≥ 60yrs) have received a total of 26 cycles of consolidation with HiDAC and crenolanib. Twelve pts (46%) have been bridged to allogeneic SCT. With a median follow up of 6 months, only three pts (all > 60yrs) have relapsed. Overall survival is shown in Figure 1.Conclusion: Crenolanib, a type I pan-FLT3 TKI, can be safely combined at full doses with cytarabine/daunorubicin or cytarabine/idarubicin induction and HiDAC consolidation chemotherapy for upfront AML therapy. A high CR rate of 88% with full count recovery was observed after one cycle of induction with an overall CR/CRi rate of 96%. HiDAC consolidation and allo SCT could be administered on schedule. Encouraging anti-leukemic activity has been observed with no relapses in FLT3 mutant AML pts <60yrs. Accrual to this trial continues. [Display omitted] [Display omitted] DisclosuresStone:Roche: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Agios: Consultancy; Amgen: Consultancy; Celator: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Novartis: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Juno Therapeutics: Consultancy; Merck: Consultancy. Eckardt:Arog: Employment, Equity Ownership.

AC220 and AraC cause differential inhibitory dynamics in patient-derived M5-AML with FLT3-ITD and, thus, ultimately distinct therapeutic outcomes.

Engrafting the bone marrow cells of a patient with M5 acute myeloid leukemia into immunocompromised mice (AM7577) resulted in serially transferrable stable AML and eventual mortality. The disease starts in the bone marrow and then expands to peripheral areas, which is typical of M5 leukemogenesis, where high leukemic burden in blood is coincident with symptoms/mortality. The leukemic cells in the mice had myeloid morphology, phenotypes, and genotypes (including the internal tandem duplication of FMS-like tyrosine kinase receptor 3 gene [FLT3-ITD]) similar to those of the original patient. Autocrine mechanisms of human granulocyte-macrophage colony-stimulating factor/interleukin-3 likely support AM7577 growth in mice. Treatment with FLT3 TKI (AC220) caused complete remission in peripheral blood, spleen, and bone, along with relief of symptoms and extended life, hinting that FLT3-ITD may be a key leukemogenic driver maintaining the disease. Interestingly, withdrawal of AC220 (high dose) did not result in relapse of disease, suggesting cure. These results, however, are in contrast to cytarabine (AraC) induction treatment: First, although AraC significantly suppresses the diseases in blood, and to a lesser degree in bone marrow and spleen, the suppression is temporary and does not prevent eventual onset of disease/death. Second, the withdrawal of AraC always resulted in rapid relapse in peripheral blood and eventually death. Our observation in this patient-derived model may provide useful information for clinical applications of the two drugs.

Clinical activity of Crenolanib in patients with D835 mutant FLT3-positive relapsed/refractory acute myeloid leukemia (AML).

7027 Background: Development of D835 is a common mechanism of resistance to most FLT3 inhibitors. Crenolanib, a type I FLT3 tyrosine kinase inhibitor, has in vitro activity against FLT3 ITD and FLT3 D835. Crenolanib trials in relapsed/refractory FLT3 mutant AML (FLT3 ITD, FLT3 D835, FLT3 ITD/D835) are ongoing (NCT01522469, NCT01657682). Methods: Clinical data on the first 19 patients (7M, 12F) with a median age of 47 years (21-81) are currently available. 6 patients were refractory and 7 had relapsed within 6 months of induction therapy. 4 had undergone prior allogeneic transplants. 11/19 had progressed after exposure to ≥1 prior FLT3 TKI (8 Sorafenib, 7 AC220, 2 PLX3397, 1 PKC412,). 3 had received 2 or more prior FLT3 TKIs. Initially, crenolanib was given at a fixed dose of 100 mg PO TID but the dose was subsequently individualized to 200 mg/m2/d given in 3 divided doses. Results: Crenolanib had a Tmax of 1.5-2 hours and a T1/2 of 8-9 hours. Median day 15 trough levels (from 11 patients) ranged from 136 ...

ETV6 /FLT3 fusion in a mixed-phenotype acute leukemia arising in lymph nodes in a patient with myeloproliferative neoplasm with eosinophilia

The ETV6/FLT3 fusion gene has been recently reported in association with myeloproliferative neoplasm with eosinophilia (MPN-Eo) and peripheral T cell lymphoma. Favorable clinical response to a targeted FLT3 tyrosine kinase inhibitor (FLT3 TKI) was noted. Here we report a novel phenotype associated with ETV6/FLT3 rearrangement, in a 38-year-old female with presentation of progressive lymphadenopathy. Lymph node biopsy showed mixed-phenotype acute leukemia (MPAL) with expression of T cell and myeloid markers. Bone marrow morphology was consistent with MPN-Eo and no evidence of MPAL. Karyotype analysis revealed 46, XX, t(12;13)(p13;q12). ETV6/FLT3 fusion was demonstrated by fluorescence in situ hybridization. This is the first report of ETV6/FLT3 rearrangement showing a phenotype of extramedullary T/myeloid MPAL arising in the setting of MPN-Eo. We suggest addition of this entity to the WHO category of “myeloid/lymphoid neoplasms with eosinophilia,” particularly given the possibility of clinical response to FLT3 TKI in MPAL, a disease usually associated with poor prognosis.

Variations in FLT3 ligand levels during the course of AML treatment.

7026 Background: FLT3 ligand (FL) is a hematopoietic growth factor expressed in many tissues. AML patients who are administered myeloablative therapy exhibit a marked and transient rise in plasma FL concentrations. Furthermore, the presence of high concentrations (1000 pg/mL) of FL impedes the efficacy of FLT3 tyrosine kinase inhibitors in vitro. However, the behavior of FL concentrations throughout the course of AML treatment remains unknown. This pilot study was undertaken to track the relationship between AML therapy and FL levels over time. Methods: Ten AML patients were enrolled in an IRB-approved procurement protocol. Blood samples were collected at weekly intervals for one year, and plasma was isolated by centrifugation. Plasma FL and stem cell factor (SCF) concentrations were measured by ELISA. Results: We observed four distinct patterns in FL fluctuations. First, in all cases where induction or consolidation chemotherapy resulted in an aplastic bone marrow (nine patients), FL concentrations rose markedly and consistently to levels &gt;1000 pg/mL following the administration of chemotherapy. Second, in three of four patients whose leukemia was refractory to induction chemotherapy, FL concentrations remained below 500 pg/mL during induction. Third, in two patients receiving the FLT3 TKI sorafenib, FL concentrations did not rise above 500 pg/mL while on this medication. Fourth, in one patient receiving the hypomethylating agent 5-azacitidine, FL concentrations remained below 100 pg/mL throughout the course of therapy. SCF concentrations did not vary throughout the course of chemotherapy. Conclusions: An “FL surge” was seen when cytotoxic chemotherapy resulted in aplasia. This FL surge was not seen with sorafenib or 5-azacitidine. In addition, the FL surge was attenuated in three patients whose leukemia was refractory to chemotherapy. These observations give rise to two new hypotheses regarding FL: 1) It is possible to maintain lower FL levels with targeted agents than with chemotherapy; and 2) Residual leukemia appears to inhibit the FL surge, providing indirect evidence of cross-talk between leukemia and the stromal microenvironment. This inhibition may be the explanation for why AML patients develop pancytopenia early in relapse.