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Differential expression analysis of primary AML cells treated with AC220 vs DMSO in endothelial cell co-culture.

ABSTRACT: Although chemotherapy can successfully induce remission in FLT3/ITD positive acute myeloid leukemia (AML) patients, many, especially those with a high ratio of the FLT3/ITD versus wild type allele (FLT3-AR), exhibit a high relapse rate, requiring hematopoietic stem cell (HSC) transplantation to increase the chance of long-term remission. As the bone marrow tumor microenvironment (TME) has been implicated in drug resistance, we reasoned that AML-TME interactions might be critical for leukemic precursor survival and drug resistance, and that targeting AML-TME interactions might be crucial to improving survival in FLT3/ITD AML patients. In this study, we demonstrate that endothelial cells (ECs), a component of the TME, and the Notch pathway plays a critical role in the protection of FLT3/ITD positive progenitors against AC220 in AML patient samples with high FLT3-AR. We have previously shown that high FLT3-AR disease is distinguished by the presence of the mutation in the least mature CD34+CD33- precursors, where leukemic stem cells (LSCs) reside. Here, we demonstrate that inhibiting Notch signaling overcomes the TME-mediated drug resistance of the LSC-enriched CD34+CD33- precursors, raising the possibility that Notch may serve as a therapeutic target for eradicating LSCs that are thought to be responsible for relapse in this high-risk AML subset.

ORGANISM(S): Homo sapiens

PROVIDER: GSE138340 | GEO | 2019/10/03

REPOSITORIES: GEO

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PRMT1-mediated FLT3 arginine methylation is required for maintenance of FLT3-ITD+ Acute Myeloid Leukemia

Project description:The presence of FLT3-ITD mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate FLT3-ITD+ leukemia stem cells (LSCs) which are potential sources of disease relapse, prompting us to ask whether FLT3-ITD protein regulates the AML LSCs survival through a kinase-independent mechanism. Here, we show that expression of PRMT1, the primary type I arginine methyltransferase, significantly increases in LSC-enriched CD34+CD38- populations relative to normal counterparts. Genetic PRMT1 depletion blocked AML CD34+ cell survival, and had more potent effects in AML cells from patients harboring FLT3-ITD. Our genome wide analysis of gene expression and PRMT1 conditional KO mouse study confirmed that PRMT1 preferentially cooperates with FLT3-ITD contributing to AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginines 972/973, and PRMT1 promoted leukemia cell growth in a FLT3 methylation-dependent manner. Moreover, effects of FLT3-ITD methylation in AML cells were in part due to crosstalk with FLT3-ITD phosphorylation at tyrosine 969 (Y969). Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following TKI (AC220) treatment, indicating that methylation occurs independently of kinase activity. Finally, in both patient-derived xenograft (PDX) and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes LSC activity and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to selectively target FLT3-ITD+ LSCs.

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Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib

Project description:Treatment with inhibitors of the receptor tyrosine kinase FLT3 are currently studied as promising therapies in acute myeloid leukemia (AML). However, only a subset of patients benefit from these treatments and the presence of activating mutations within FLT3 can predict response to a certain extent only. AC220 (quizartinib) is an example of a potent FLT3 inhibitor1 that was studied in a recent phase II open-label study in patients with relapsed/refractory AML. The complete remission rate (including CRp and CRi) in FLT3-ITD-positive patients was 54% (50/92) and the corresponding partial remission rate (PR) was 17% (16/92)2 Thus, although the FLT3-ITD mutation status correlates with response, the error rate in stratification of patients into responders and non-responders is high, as still 29% of the FLT3-ITD-positive patients failed to respond. Exclusion of FLT3-ITD-negative patients from AC220 treatment also seems critical, as the total response rate (CRþPR) in FLT3-ITD-negative patients is substantially lower (41%, 17/41). As AC220 is a tyrosine kinase inhibitor, we hypothesized that investigating phosphorylation-based signaling on a system-wide scale in AML cells allows for identification of markers enabling more accurate prediction of therapy response as compared to commonly used genetic markers. Hence, we applied quantitative mass spectrometry to decipher a multivariate phosphorylation site marker, which we refer to as phosphosignature, in patient-derived AML blasts that might be useful as predictive biomarkers for AC220 treatment.

2017-05-10 | PXD006475 | Pride

Alkynyl nicotinamides with antileukemic activity to treat poor prognosis AM

Project description:FLT3 activating mutations cause myeloproliferative neoplasms by deregulating hematopoietic progenitor cell growth, and acute myeloid leukemia is on the rise. Investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop inherent and acquired resistance to FLT3 targeted therapy. FLT3 inhibitor resistance in AML is dependent on co-occurring mutations, parallel survival pathways, and/or subsequent FLT3-ITD mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options. We identified two novel naphthyridine-based FLT3 inhibitors (HSN608 and HSN748) that specifically target FLT3-ITD at sub-nanomolar concentrations and are effective against drug-resistant secondary mutations. In the current study, we evaluated these compounds antileukemic activity against FLT3-ITD and gatekeeper mutations in drug-resistant AML, relapsed/refractory AMLs with FLT3 mutations, and in vivo mouse models with combinations of epigenetic mutations TET2 with FLT3-ITD, and AML patients PDX. In these model systems, we demonstrate that HSN748 outperforms the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3-ITD.

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2017-01-30 | GSE81420 | GEO

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Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 AML Cells

Project description:Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, application of FLT3 small molecule inhibitors such as Sorafenib has resulted only in partial and transient clinical responses in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with Sorafenib in addition to standard therapy. Here, we studied the Sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 µM, whereas FLT3-ITD+ cells (MOLM-13, MV4;11) were found more sensitive to Sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also Sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in Sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4;11 cells Sorafenib suppressed mTOR signalling by inhibiting direct inhibition of FLT3. In MONO-MAC-1 cells Sorafenib inhibited the MEK/ERK pathway by inhibition of the RET tyrosine kinase. These data suggest that the FLT3 status in AML patients might not be the only predictive factor for a response to Sorafenib.

2017-05-02 | PXD004442 | Pride

GRP94 rewires and buffers the FLT3-ITD signaling network and promotes survival of acute myeloid leukemic stem cells

Project description:Internal tandem duplications in the tyrosine kinase receptor FLT3 (FLT3-ITD) are among the most common lesions in acute myeloid leukemia (AML) and there exists a need for new forms of treatment. Using ex vivo drug sensitivity screening, we found that FLT3-ITD+ patient cells are particularly sensitive to HSP90 inhibitors. While it is well known that HSP90 is important for FLT3-ITD stability, we found that HSP90 family members play a much more complex role in FLT3-ITD signaling than previously appreciated. First, we found that FLT3-ITD activates the unfolded protein response (UPR), leading to increased expression of GRP94/HSP90B1. GRP94 rewires FLT3-ITD signaling by binding and retaining FLT3-ITD in the ER, where it aberrantly activates downstream signaling pathways. Second, HSP90 family proteins protect FLT3-ITD+ AML cells against apoptosis by alleviating proteotoxic stress, and treatment with HSP90 inhibitors results in proteotoxic overload that triggers UPR-induced apoptosis. Importantly, leukemic stem cells are strongly dependent upon HSP90 for their survival, and the HSP90 inhibitor ganetespib causes leukemic stem cell exhaustion in mouse PDX models. Taken together, our study reveals a molecular basis for HSP90 addiction of FLT3-ITD+ AML cells and provides a rationale for including HSP90 inhibitors in the treatment regime for FLT3-ITD+ AML.

2020-09-02 | PXD009423 | Pride

High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive in vitro kinase assay substrates

Project description:Abnormalities in the FLT3 signaling pathway play an integral role in AML disease relapse and drug resistance. Developing new and specific FLT3 tyrosine kinase inhibitors for use in combination to induction therapy is an important step to reduce disease relapse and achieve clinical remission. To develop potent FLT3 TKI requires sensitive in vitro assay that depended on efficient FLT3 artificial substrates, which there are none reported for FLT3 WT and kinase variants. The kinase assay linked with phosphoproteomics was applied as a high throughput technique to increase the known FLT3 kinase substrates (WT, ITD and D835Y) that were used to identify the FLT3 kinase variant’s preferred kinase sequence using the KINATEST-ID substrate predictive pipeline. The identified substrate sequence was used to synthesize and validate pan-FLT3 artificial substrates to monitor in vitro kinase activity in the presence of clinically relevant FLT3 TKI.

2019-01-07 | PXD011478 | Pride

Molecular variability of FLT3/ITD mutants and their impact on the differentiation program of 32D cells

Project description:FLT3 is the most frequently mutated gene in AML â up to 40% of AML harbor an activating mutation within FLT3 gene. Though AML is a relatively rare disease, such a high mutability rate, as observed with FLT3 gene, is striking. To elucidate the molecular background of this phenomenon, we have established nine unique FLT3/ITD - carrying 32D cell lines and a set of controls, and subjected them to whole genome expression analysis and 2DE LC/MS proteomics. Data obtained on this so far largest set of ITD mutants indicates that in comparison to the wild type FLT3 expressing 32D cells and transduction controls, FLT3/ITD positive cells exhibit less mature expression profiles resembling ST-HSC and MkEP/CMP/LMPP progenitors. We hypothesize that FLT3/ITD might contribute not only to the proliferative advantage of FLT3/ITD positive cells, but also to their reprogramming towards less differentiated stages, thus strengthening their malignant properties. This finding might explain the pronounced mutation rate of the aberrantly expressed FLT3 gene in AML, and, also, the inferior prognosis of FLT3/ITD positive AML patients. Moreover, the microarray data has revealed biological differences among individual ITD variants â a finding supporting the recent clinical data on the prognostic impact of the size of individual ITDs. Keywords: genetic modification Nine stable 32D cell lines harboring unique human FLT3/ITD mutants, two parental 32 cell lines, two 32D stable cell lines harboring cloning vector only, and two 32D cell lines stably expressing human wild type FLT3 were subjected to the microarray analysis.

2010-04-08 | E-GEOD-12766 | biostudies-arrayexpress

Sorafenib treatment of FLT3-ITD+ acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent non-responsiveness associated with a D835 mutation

Project description:Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% acute myeloid leukemias (AML) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD+ AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow (BM) myeloblasts after 27 (range 21–84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54–287) days but the FLT3 and downstream effectors remained suppressed. Four pairs patients (before sorafenib treatment and after sorafenib relapse), total eight samples from four patients at the two time-points were subjected to microarray analysis. Gene expression profiling showed that leukemia cells which have become sorafenib resistant expressed a number of genes including ALDH1A1, JAK3 and MMP15, whose functions were unknown in AML. NOD/SCID mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain (TKD) mutations at D835 were identified in leukemia initiating cells (LIC) from samples before sorafenib treatment. LIC bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors. RNA from CD33+CD34+ myeloblasts at pre-sorafenib-treatment and post-sorafenib-relapse were collected and subjected to microarray analysis

2012-02-18 | E-GEOD-35907 | biostudies-arrayexpress

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