Wt FLT3 Research Articles

Preliminary safety, efficacy and molecular characterization of emavusertib (CA-4948) in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) with FLT3 mutation (FLT3m).

6539 Background: AML is a heterogenous disease and exhibits a dynamic mutational landscape as the disease progresses. Internal tandem duplication (ITD) of FLT3 is considered an acquired late-event mutation and is associated with a poor prognosis in AML. Emavusertib is a potent oral inhibitor of both IRAK4 and FLT3, conferring potential dual efficacy advantages compared to other IRAK4 or FLT3 inhibitors as single agents. IRAK4 is upregulated during anti-FLT3- or other cytotoxic therapies, which drives a resistance pathway of early relapse and progression. Methods: The safety, clinical activity, and potential biomarkers of emavusertib in R/R AML and higher-risk myelodysplastic syndrome are being investigated in the ongoing open-label, Phase 1/2a TakeAim Leukemia trial (NCT04278768). Next generation sequencing (NGS) of 68 genes was performed on genomic DNA from bone marrow or peripheral blood mononuclear cells at baseline and on treatment. Here, we present preliminary safety, efficacy data and molecular characterization in a subset of enrolled AML patients who carried FLT3m at baseline and were treated with emavusertib monotherapy. Results: In this study, we treated 11 R/R AML patients with FLT3m at dose levels of 200-400 mg BID. The median number of prior anti-cancer therapies was 2 (range 1-6). 8 of 11 patients had prior exposure to FLT3 inhibitors. Treatment-related adverse events (TRAEs) Grade ≥ 3 were reported in 2 of 11 (18%) patients. All responders were dosed at the Recommended Phase 2 Dose (RP2D) of 300 mg BID and with < 3 lines of prior therapy. In addition, responders demonstrated more than 90% bone marrow blast reduction compared to baseline. The most common co-mutations in this subset of patients included RUNX1, NRAS, and TET2. Emavusertib treatment significantly decreased the variant allele frequency of these mutations. FLT3-ITD levels were decreased or became undetectable in responders. Additional corelative analysis between bone marrow blast counts and mutation status indicates that AML patients with FLT3m demonstrated increased bone marrow blast reductions (P≤0.05) on emavusertib treatment, when compared to FLT3 WT AML patients. Conclusions: Emavusertib has a favorable safety and tolerability profile in pretreated AML patients with FLT3m and demonstrated monotherapy anti-cancer activity in patients with FLT3m, including patients who have progressed on prior FLT3 inhibitors. Mutational profiles are suggestive of disease-modifying activity of emavusertib. We will present updated safety and efficacy data, including data for patients who proceeded to stem cell transplant. Enrollment in this ongoing trial is continuing at RP2D in patients with < 3 lines of prior anti-cancer therapies. Clinical trial information: NCT04278768 .

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract 4413: Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development

Abstract Acute myeloid leukemia (AML) is a complex disease with highly individualized treatment plans for patients. Mostly these are based on genetic and phenotypic markers of the disease, but the patient’s overall condition is playing a significant role as well. To develop innovative drugs for a broad patient cohort, it is essential to understand the biological implications of these differences and to model them preclinically. In this study, we are assessing the utility of EpiCypher’s CUT&Tag-FFPE platform to characterize the epigenome of AML PDX samples. CUT&Tag-FFPE is an epigenome profiling assay that is compatible with Formalin-Fixed Paraffin-Embedded (FFPE) samples, enabling robust genome-wide profiling of histone modifications from limited clinical samples. We derived PDX model LEXFAM 2799 (NPM1 wt; FLT3 wt, t(8;21)) and LEXFAM 2966 (NPM1 mut; FLT3 wt) from treatment naïve patients and established in immunocompromised NSG mice. LEXFAM 2799 is a fast-growing model with passaging times of 33 days, whereas LEXFAM 2966 displays a passaging time of 140 days. We next treated with Standard of Care (SoC) agents Decitabine, Cytarabine and all-trans-retinoic acid (ATRA): LEXF 2966 was highly sensitive towards Decitabine leading to complete remission. Cytarabine induced a partial remission and ATRA had only minor effect on tumor growth. In contrast LEXFAM 2799 was resistant towards treatment with ATRA and showed only partial remission under treatment with Cytarabine or Decitabine. Using the CUT&Tag-FFPE platform we are analyzing FFPE slides from the above mentioned two AML PDX models to investigate differences in the genome-wide histone profile that relate to the different growth behavior as well as sensitivity towards SoC treatment. The goal of these studies is to identify tumor model specific profiles that correlated specifically with the sensitivity towards the hypomethylating agent Decitabine. In addition, we plan to analyze additional untreated as well as treated AML PD models to validate the identified signatures confirming them as predictive biomarkers for sensitivity towards current standard of care and prospectively as well innovative compounds. Citation Format: Alysha Simmons, Eva Oswald, Georg Kuales, Michael Keogh, Vishnu U. Kumary, Eva Brill, Martis W. Cowles, Bryan Venters, Julia B. Schueler. Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4413.

Symptomatic Patients with Hyperleukocytic FLT3-ITD Mutated Acute Myeloid Leukemia Might Benefit from Leukapheresis.

We aimed to identify subsets of patients who benefit from emergency LA and to establish a therapeutic algorithm for AML patients with hyperleukocytosis. In this single-center retrospective cohort study, a total of 20 consecutive patients underwent LA because of their clinical symptoms. Overall survival (OS) analysis was conducted using the Kaplan-Meier plot method. Univariate and multivariate analyses (using multiple logistic regression) were performed. At the time of diagnosis, all patients received a standard diagnostic workup for AML including FLT3-ITD mutational analysis. FLT3-ITD mut AML patients receiving LA had a median OS of 437 days (range 5-2379 days) with a corresponding 14-day survival of 92.3%, while FLT3 wt AML patients displayed a significantly lower median OS of only 5 days (range 1-203 days) with a corresponding 14-day survival of 14.3% (p = 0.0001). Among patients with clinical symptoms of leukostasis, the subset of FLT3-ITD mut AML patients showed a better outcome with lower early mortality after emergency LA. Based on these observations, we established a therapeutic algorithm for AML patients with hyperleukocytosis.

Quantum-First (Q-F): Clinical Bridging Study for FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD) Companion Diagnostic Development

Background Q-F (NCT02668653) showed that the highly potent, selective, type 2 FLT3 inhibitor quizartinib (Q) + standard chemotherapy ± transplantation, followed by Q monotherapy for ≥36 cycles, reduced the relative risk of death by 22.4% vs placebo (P) in newly diagnosed (nd) FLT3-ITD+ AML, with HR of 0.776 and P value of 0.0324 (PMID: 37116523). In Q-F, FLT3-ITD mutation status was determined using a FLT3-ITD mutation detection clinical trial assay (CTA) validated under design control by Navigate BioPharma Services, Inc. We describe the results of the bridging study, aimed to show agreement between the CTA & the LeukoStrat CDx FLT3 Mutation Assay (CDx, by Invivoscribe) in FLT3-ITD+ pt selection and to determine if Q efficacy (overall survival [OS]) was maintained in nd FLT3-ITD+ AML pts from Q-F, if pts had been selected using the CDx. Methods In both CTA & CDx, DNA, extracted from bone marrow (n=884 each) or peripheral blood (n=139 each), was amplified via PCR and amplicons were detected via capillary electrophoresis. A sample was considered CTA+ if the variant allele frequency ( FLT3-ITD/total FLT3) was ≥3% and CDx+ if the signal ratio (SR; FLT3-ITD/ FLT3 WT) was ≥0.05. The agreement between CTA & CDx was based on evaluating CTA+ & CTA− samples with the CDx assay. A primary analysis included the CDx detected (CDx+ & CDx−) and the CDx invalid results. A secondary analysis used CDx+ and CDx− results only. To establish agreement between CDx & CTA, positive % agreement (PPA) and negative % agreement (NPA) were determined using CTA results as reference for the agreement analysis set (AAS). Concordance was established if the lower bounds of the 95% CIs for both PPA & NPA exceeded 90% for the analysis that included the invalid CDx results. Median OS in the subgroups was calculated based on Kaplan-Meier estimates. Stratified Cox proportional hazards regression model was used to estimate HRs, 95% CI, and P value. Results Full analysis set (N=3468) included all Q-F screened CTA+ pts (n=863), all screened CTA− pts (n=2556), and pts with unknown CTA status not eligible for randomization due to other criteria (n=49). Of these, 1032 pts formed the primary analysis set (PAS), including all pts randomized in Q-F with samples available for CDx testing (N=513: Q, n=254; P, n=259) and a randomly selected subset of CTA− pts (n=519). The ascertainment rate was 95.2% (513/539), as 26 of the 539 pts randomized in Q-F were excluded from the bridging study. Within the PAS, 3 samples were not tested by CDx due to insufficient volume/DNA amount. The AAS (N=1029: CTA+, n=513; CTA−, n=516) included pts in the PAS with valid CTA results and tested with CDx. In the AAS, 6 samples (3 CTA+, 3 CTA−) did not yield valid CDx results, resulting in 1023 CDx-evaluable total pts (CTA+, n=510; CTA−, n=513). Among 510 CTA+ samples, 483 were CDx+. Among 513 CTA− samples, 513 were CDx−. Therefore, 996 samples yielded concordant results, 27 samples yielded discordant results, and 6 samples did not yield a valid CDx result for comparison. Point estimates of PPA & NPA were 94.2% & 99.4%, respectively, with invalid CDx results included in the calculation, and 94.7% & 100%, respectively, without invalid CDx results. The lower bounds of the 95% CIs were all above the corresponding acceptance criterion of 90% for PPA & NPA (Table 1). In Q-F, the prevalence of CTA+ was 24.9% among screened pts (863/3468), whereas in the bridging study, 49.9% (510/1023) of pts were CTA+: this enrichment in CTA+ pts could lead to a biased estimate of the agreement between CDx & CTA when using CDx as reference. The positive predictive value (PPV) and negative predictive value (NPV) of the CDx adjusted for this enrichment ± invalid CDx results, showed that the lower bounds of the 95% CIs were all >95% (Table 1). The efficacy OS analysis in the intent-to-treat (ITT) CDx+ population (ITT CDx+=CTA+ & CDx+; N=483: Q, n=242; P, n=241) demonstrated a clinically relevant OS improvement with Q (median OS of 29.4 months) vs P (median OS of 14.8 months), resulting in 14.6 months prolongation of median OS, with an HR of 0.794 (95% CI 0.621-1.014), corresponding to a 20.6% reduction in relative risk of death (Figure 1). Conclusions This study showed 1) agreement between CDx & CTA in identifying nd FLT3-ITD+ AML pts and 2) that OS benefit provided by Q in the ITT CDx+ population is comparable with the OS benefit in the ITT population of Q-F. The LeukoStrat CDx FLT3 Mutation Assay aids in assessing AML pts for Q therapy.

Overexpression of the Signaling Integrator Gab2 Accelerates AML Development in Mice with Dnmt3a R878Hand Npm1 cA Mutations

NPM1 and DNMT3A are among the most commonly mutated genes in Acute Myeloid Leukemia (AML), affecting 25-30% of patients. Importantly, these mutations co-occur much more frequently than expected by chance, with ~15% of AML samples containing mutations in both genes.This synergy is recapitulated in mouse models, where mice with either Dnmt3aR878H or Npm1cA mutations develop leukemia with a long latency and low penetrance, whereas mice with both Dnmt3aR878H and Npm1cA mutations develop leukemia in 8-12 months with nearly 100% penetrance (Dovey et al., Blood, 2017; Loberg et al., Leukemia, 2019).To better understand the mechanisms underlying this synergy, and the process of leukemic transformation, we have characterized pre-leukemic bone marrow and 9 independent spontaneous AMLs that have arisen in these mice. These spontaneous AMLs rapidly cause fatal leukemias in secondary recipients; whole genome sequencing of all tumors revealed one or more human AML-like cooperating mutations in each, including canonical mutations in Ptpn11 (E69K, A72V and S506L), an activating KitD818Y mutation, a Cbl exon 9 deletion, a CblY369H mutation,and an amplification of Flt3 associated with >20 fold overexpression of WT Flt3. Characterization of the pre-leukemic transcriptomes and methylomes demonstrated little change in the pre-leukemic state for several months, followed by dramatic and canonical shifts in transcription and methylation after transformation. Remarkably, 8 of the 9 AMLs contained an amplification of the entirety of murine chromosome 7 as the sole structural variant in each genome. We analyzed panel sequencing data from a set of published AMLs arising in this model (Loberg et al., Leukemia, 2019), and also exome sequencing data from a different Npm1 cA driven tumor model (Dovey et al., Blood, 2017); these AMLs also developed complete or partial amplification of chromosome 7 in 7 of 8 and 10 of 15 cases, respectively. Through integration of these datasets, we identified a minimally amplified 8.9 Mbp region on chromosome 7, containing 209 genes, including two candidate genes ( Gab2, Pak1) that show overexpression in both murine and human AMLs with DNMT3A and NPM1 mutations. Retroviral constructs expressing each cDNA (with an IRES-GFP tag) were transduced into lineage depleted bone marrow cells from preleukemic WT, Dnmt3aR878H, Npm1cA or Dnmt3aR878H x Npm1cA mice, and transplanted into recipient mice. Overexpression of GAB2 (but not “Empty Vector” or PAK1) induced significant expansion of hematopoietic cells from the Dnmt3aR878Hx Npm1cAmice (and to a lesser extent, from Npm1cA mice),but not Dnmt3aR878H mice or WT mice ( Figure 1A), suggesting that the mutations in Npm1 and Dnmt3a synergize to account for this phenotype. Strikingly, overexpression of GAB2 in Dnmt3aR878Hx Npm1cAbone marrow led to development of AML in 5 of 5 engrafted mice in less than 5 months (p<10 -6 compared to untransduced marrow; Figure 1B). 0 of 3 engrafted mice with “Empty Vector” (GFP only) in Dnmt3aR878Hx Npm1cAbone marrow and 0 of 3 engrafted mice with overexpression of PAK1 in Dnmt3aR878Hx Npm1cAbone marrow developed leukemia during this 5 month period. Taken together, these data suggest that AML-associated mutations in DNMT3A and NPM1 allow for the selective expansion of cells overexpressing GAB2 as an important step towards leukemic transformation. In human myeloid neoplasms, amplification of chromosome 11q (containing human GAB2) is a recurrent cytogenetic event, observed in 1.5% of cases (Papaemmanuil, NEJM, 2016), and expression of GAB2 across AML samples containing NPM1 mutations is increased ~50% compared to healthy CD34+ cells or promyelocytes. GAB2 is known to bind to GRB2 and SHP2/PTPN11 to facilitate signaling from receptor tyrosine kinases (including FLT3) to downstream pathways (including RAS/RAF/MEK/ERK, PI3K and AKT, in some contexts). These data nominate GAB2 overexpression as a factor that may be relevant for the progression of founding clones with DNMT3A and NPM1 mutations to overt AML. Additional studies to define its protein interactome in preleukemic cells, its mechanism of action, and how leukemia-causing mutations in DNMT3A and NPM1 shape the fitness landscape to select for GAB2 overexpression are underway.

Easyab Unravels Oncokinase Driven Regulation of Splicing Factors in AML

Acute myeloid leukemia (AML) is the most commonly diagnosed leukemia in adults. Gene mutations involved in various cellular functions are known to cause AML. A large proportion of AML is driven by aberrant activation of kinases and signal transducers such as FLT3, cKIT, PTPN11, JAK2, KRAS, NRAS, and to a lesser extent, JAK1, JAK3, and CSF3R. Treatment for AML has remained the same in the past five decades. Recent developments led to the availability of targeted therapies and enabled the practice of additional drugs like tyrosine kinase inhibitors. However, drug resistance and tolerance mechanism remain a challenge to finding a cure for the disease. Though we can identify and target kinase mutations individually, we have yet to learn how these mutations, like FLT3-ITD, change the cell's intrinsic signalling environment and cooperate with other oncogenes. Understanding these signalling behaviors is crucial for effective treatment outcomes and avoiding the development of drug resistance and tolerance mechanisms. Protein post-translational modifications (PTM) such as phosphorylation, ubiquitination, methylation, control the activity, fate, and spatial location of the proteins, thereby controlling processes such as signal transduction. Mass spectrometry-based proteomics offers unbiased profiling of PTMs. However, comprehensive analyses of PTMs require conventional antibody-based enrichment approaches and large starting materials, laborious, low throughput, cost prohibitive, and time-consuming, thus limiting the application to cell line-based studies. It also limits application in exploring unbiased-global cell signaling in patient samples. To overcome these limitations, we developed a loss-less sample preparation method named ‘EasyAb’. We show that our short, high throughput and low-cost approach enables capturing PTM peptides from very small inputs. As proof of principle, we enriched tyrosine-phosphorylated peptides from Molm13 and MV4-11 cell lines expressing FLT3-ITD kinase. Enrichment resulted in the ultra-deep identification of 4,416 tyrosine phosphosites, double the number of sites found in the conventional protocols, and took only one day of processing. About 40 % of the tyrosine phosphorylated proteins enriched are membrane proteins, including seven tyrosine phosphorylation sites on FLT3 kinase, of which FLT3 pY401 is a novel site discovered. To test the applicability of the EasyAb protocol in patient samples, we performed phosphotyrosine profiling of AML samples expressing FLT3 WT (n=9) and FLT3-ITD (n=5) mutation. EasyAb enrichment for phosphotyrosine peptides resulted in identifying 6,339 phosphotyrosine sites, of which 2300 sites were quantified. Comparing FLT3WT and ITD samples revealed more than 500 phosphosites significantly regulated. The significantly changing sites include several known signalling molecules such as STAT5, SYK, BTK, GAB2, GRB2, and PTPN11. The data also revealed differential tyrosine phosphorylation of proteins involved in various cellular functions such as autophagy, apoptosis, cell cycle, DNA damage repair proteins, phosphatases, and kinases. Interestingly, we also uncovered a class of proteins belonging to mRNA processing and splicing pathways distinctly tyrosine phosphorylated in FLT3-ITD samples. It is known that splicing factor mutations drive leukemia. Conversely, little is known about the regulatory mechanism of splicing factors by singling in cancer. To further investigate FLT3-ITD signalling regulation of splicing factors, we treated Molm13 and MV4-11 cells with Quizartinib and profiled tyrosine phosphorylation. Inhibition of FLT3-ITD lead to downregulation in tyrosine phosphorylation of several splicing factors. We have previously reported that splicing factor phosphorylation by JAK2 kinase plays a crucial role in Jak inhibitor persistence phenotype. Importantly, FLT3-ITD-mediated tyrosine phosphorylation-based regulation of splicing factors is unknown, and this process could be crucial for FLT3-ITD-mediated tumorigenesis. The functional significance of this novel regulatory mechanism is being studied. In conclusion, we have developed an effective tool for studying post-translational modifications that require antibody-based enrichment (tyrosine phosphorylation, ubiquitination, methylation, acetylation, and sumoylation) relatively quickly in primary patient samples.

Autologous stem cell transplantation in adult patients with intermediate-risk acute myeloid leukemia in first complete remission and no detectable minimal residual disease. A comparative retrospective study with haploidentical transplants of the global committee and the ALWP of the EBMT

In patients with acute myeloid leukemia (AML) of intermediate-risk (IR) in first remission (CR1) with no measurable residual disease (MRD negative), the choice of the best consolidation is questionable. 1122 adult patients from 196 centers, transplanted in 2010-21 were analyzed: 547 received an autologous stem cell transplantation (ASCT) and 575 a Haploidentical donor transplant. Because of a significant interaction, comparisons were done separately for patients with wild-type FLT3 (FLT3-wt) and FLT3-ITD mutation (FLT3-ITD). In FLT3-wt patients, haploidentical transplants had two year lower relapse incidence (RI) (16.9% versus 32.6%; HR = 0.40, p < 0.001), higher NRM higher (17.2% vs 3.5%; HR = 7.02, p < 0.001), similar LFS (65.9% vs 63.8%; p = 0.37) and lower OS (73.2% vs 80.6%; HR = 1.69, p = 0.018). In FLT3-ITD patients, haploidentical transplants had two year lower RI (8.2% vs 47.8%; HR = 0.14, p < 0.001) higher NRM (20.2% vs 5.6%; HR = 3.43, p = 0.002), better LFS (71.5% vs 46.6%; HR = 0.53, p = 0.007) and similar OS (73.5% vs 61.9%; p = 0.44). In IR AML patients with FLT3-wt in MRD negative CR1, autologous stem cell transplantation is a valid option, while in patients with FLT3-ITD, haploidentical transplant is better. Whether autologous transplantation is superior to chemotherapy in FLT3-wt patients and the role of maintenance therapy with FLT3 inhibitors remain to be studied.

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

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Anisomycin is active in preclinical models of pediatric acute myeloid leukemia via specifically inhibiting mitochondrial respiration.

The poor outcomes in acute myeloid leukemia (AML) necessitate new treatments. In this work, we identified that anisomycin is a potential selective anti-AML candidate, particularly for those with FLT3-ITD mutation. We found that anisomycin potently inhibited proliferation and induced apoptosis in multiple AML cell lines. Anisomycin was effective in targeting progenitor cells isolated from all tested pediatric AML patients, while sparing normal counterparts. Using AML xenograft mouse models, anisomycin exhibited inhibitory effect on tumor growth throughout the whole duration without causing toxicity in mice. The combination of anisomycin with standard of care drugs is synergistic and selective in AML cell culture system and mouse model. In addition, FLT3-ITD cells were more sensitive to anisomycin than FLT3 WT cells. Mechanistic studies revealed that anisomycin acted on AML in a p38-independent manner. We found that anisomycin decreased mitochondrial respiration by disrupting complex I activity, leading to intracellular oxidative stress. AML ρ0 cells that lack of mitochondrial respiration exhibited resistance to anisomycin. Finally, we showed that mitochondrial biogenesis contributes to differential sensitivity of FLT3-ITD and FLT3 WT cells to anisomycin. Our work is the first to systematically demonstrate that anisomycin is a useful addition to the treatment armamentarium for AML. Our findings highlight the therapeutic value of mitochondrial respiration inhibition in AML patients harboring FLT3-ITD mutation.

Factors Predicting Response and Resistance to Midostaurin in FLT3 Positive and FLT3 Negative AML in 483 Primary AML Patient Samples

Introduction: Midostaurin is an FDA approved, multi-target tyrosine kinase(TK) inhibitor shown to inhibit the FLT3 kinase and have therapeutic benefit in FLT3 mutated AML especially FLT3-ITD mutations (FITD+). However, midostaurin has also shown therapeutic response in select FLT3-ITD negative (FITD-) AML patients likely due to inhibitory effects on other TKs .We analyzed primary AML patient samples from our ‘Beat AML’ database to look at disease related factors, aside from FLT3 status, that may influence midostaurin drug sensitivity and affect patient outcomes.Methods: We collected peripheral blood and bone marrow samples from 483 newly diagnosed AML patients (348 FITD+, 135 FITD-), extracted mononuclear cells and plated them on a Midostaurin dose curve, without growth media or feeder cells. Metabolic viability was assessed after 72-hours and drug response was summarized with an area under curve (AUC) metric on a 0-100 scale. We annotated these samples for known chromosomal translocations and somatic mutations in leukemia-related genes and performed association-based analyses to identify genomic predictors of midostaurin drug sensitivity.Results: FLT3-ITD was predictably the strongest association with midostaurin-response, so further analysis was subsetted into FITD+ and FITD- samples (Figure A). For our analysis, we included FLT3-TKD mutant samples in the FITD- cohort as we found no significant difference between FLT3-TKD status and non-FLT3 mutated samples.Within FITD- samples, we found inv(16) Core Binding Factor AMLs demonstrated increased sensitivity to midostaurin (p < 0.05) . We investigated the frequency of c-Kit mutations in inv(16) AMLs, given Midostaurin's activity against c-Kit, but did not observe significant differences in drug response between any combination of inv(16) and c-Kit mutational status due to a relatively small sample size.(Fig B) Further, among all FITD- samples evaluated, we found mutations in KRAS and TP53 to be predictive of midostaurin resistance (p < 0.05, Fig B). This relationship was also observed through analysis of publically available cell line drug screens within the Genomics of Drug Sensitivity in Cancer (GDSC), where mutations in NRAS and TP53 suggested Midostaurin resistance.In FITD+ AML samples, we found the FLT3-ITD mutant allele fraction (MAF) to strongly correlate with the extent of Midostaurin sensitivity. FLT3-ITD MAF was significantly higher in Midostaurin-sensitive samples compared to resistant samples (p < 0.001)(Fig C). Additionally, we observed an increase in Midostaurin efficacy (p < 0.05) in FLT3-ITD positive(15;17) APL,a subset of APL with relapse rates 15-times higher than FLT3 WT APL (Breccia et. al. Haematologica 2013) (Figure C). Further, we found that neither NPM1 or FLT3-TKD status conferred additional sensitivity to Midostaurin. There was no significant difference in AUC between FLT3-ITD and NPM1 double-mutant samples and FLT3-ITD alone, and similarly no significant difference between FLT3-TKD and FLT3 WT samples.Discussion: With the approval and clinical use of Midostaurin in FLT3 positive AML patients it is important to find and validate factors influencing Midostaurin efficacy. Data from the RATIFY clinical trial has shown Midostaurin improved overall survival in patients with both high and low FLT3-ITD allelic ratios (Stone et. al. N Eng J Med 2017). We show that the ex vivo Midostaurin response is correlated with FLT3-ITD status and its MAF but not significantly with the FLT3-TKD mutation. Our data suggests that FITD+ APLs are responsive to this FLT3-inhibitor thus offering the potential of combination therapy and improved outcomes with current APL regimens.We also demonstrate that Midostaurin shows efficacy in FLT3-ITD negative patient samples, illustrating the multi-targeted nature of the inhibitor. Within these samples,inv 16 predicted Midostaurin sensitivity regardless of c-Kit mutational status. Additionally, mutations in TP53 and the Ras-pathway predict Midostaurin resistance (these mutations rarely co-occurred with FLT3-ITD in our cohort, thus limiting their impact in FLT3-ITD positive cases). Additional in vitro and clinical data is necessary to determine whether these findings should influence treatment decisions.Newer FLT3 inhibitors in clinical trials need to be characterized in a similar manner so as to efficiently target subsets of FLT3 mutated AML. [Display omitted] DisclosuresTyner:Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Janssen Pharmaceutica: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Syros: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Agios Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; Leap Oncology: Consultancy. Borate:Novartis, Jazz: Consultancy, Speakers Bureau.

Differential Impact of Conditioning Intensity on Post-Transplant Outcome of FLT3 -Mutated and FLT3 Wild Type AML in First Remission, a Report from Taiwan Bone Marrow Transplant Registry (TBMTR)

AML with FLT3 -internal tandem duplication mutation (FLT3-ITD) has a very high relapse rate when patients were treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) as soon as possible once remission is achieved is generally recommended. Nevertheless, the best conditioning regimen, specifically the myeloablative (MAC) or reduced intensity conditioning (RIC), for FLT3-ITD AML remains unknown. Of the two largest series reported so far, vast majority of the patients received myeloablative regimen (100% in EBMT report, 87% in CIBMTR report). In this study, we utilize the Taiwan Bone Marrow Transplant Registry (TBMTR) database to analyze the impact of conditioning intensity on the post-transplant outcome of FLT3 -mutated and FLT3 wild type AML.TBMTR is a research collaboration affiliated to the Taiwan Society of Blood and Bone Marrow Transplantation. It comprises all the 14 transplantation centers in Taiwan that contribute detailed information on HSCT. In this study, only adults aged ≥ 18 years with a diagnosis of de novo AML who received allogeneic peripheral blood stem cell transplantation (PBSCT) at the first complete remission (CR1) and with known FLT-ITD mutation status in the registry were included. Patient characteristics and transplant outcome following MAC or RIC allogeneic PBSCT were compared. Kaplan-Meier estimates were used to calculate the probability of LFS and overall survival (OS). Cumulative incidence of relapse and non-relapse mortality (NRM) was calculated from the time of transplant to the time of event. NRM was considered a competing risk for relapse and vice versa. Multivariable analyses for LFS and OS were performed using Cox proportional hazards model.146 patients who met the eligibility criteria were enrolled for analysis. The median follow-up of survivors was 21 months (range, 1-73 months). Of the 146 patients, 49 (33.6%) were positive and 97 (66.4%) were negative for FLT3-ITD mutation. 32.7% and 35.1% of FLT3-ITD mutated and FLT3 WT AML received RIC regimen respectively. FLT3-ITD mutated patients had significantly higher WBC count (51.7 x 109/L) and more normal (64.9%) karyotype at diagnosis comparing to patients without FLT3 mutation (11.8 x 109/L WBC count and 42% normal karyotype). Patients who received RIC regimen were significantly older than patients who received MAC regimen (53.196±12.107 vs 38.327±11.788, p<0.001, for FLT3-ITD mutated AML; 52.084±12.533 vs 39.181±10.704, p<0.001, for FLT3 WT AML).In multivariable analysis, FLT3 mutation status did not negatively impact the transplant outcome in term of both overall survival (OS) and leukemia free survival (LFS). However, RIC regimen is associated with a significant poor LFS (HR 1.754, p=0.0476) and higher relapse rate (HR 2.184, p=0.015). The impact of conditioning intensity is even more prominent in patients of FLT3 WT AML (Figure 1, left, p=0.0468 for LFS, log rank test). For FLT3-ITD mutated AML, the LFS and relapse rate were almost the same after either MAC or RIC conditioning (Figure 1, right, p=0.768 for LFS, log rank test). For NRM and cumulative incidence of acute and chronic GVHD, the differential impact of conditioning intensity was not seen in FLT3-ITD mutated AML and FLT3 WT AML.In conclusion, conditioning intensity has a differential impact on post-transplant outcome of FLT3-ITD mutated AML and FLT3 WT AML. Reduced intensity conditioning is associated with a lower LFS and higher relapse rate for FLT3 WT AML. For patients of FLT3-ITD mutated AML, RIC is just as good as MAC, even though patients who received RIC were almost 15 years older than patients who received MAC. RIC allogeneic HSCT should be a viable option for elderly or unfit AML with FLT3-ITD mutation. [Display omitted] DisclosuresYao:Celgene International Sàrl: Research Funding. Tang:Celgene International Sàrl: Research Funding.

Activity of the multikinase inhibitor TP‐0903 in RAS mutant acute myeloid leukemia

Clonal heterogeneity and co‐occurring mutations contribute to drug resistance in AML. Leukemia samples from patients progressing on gilteritinib, a FLT3 inhibitor, were selected for RAS mutant clones, which are known to contribute preclinically to resistance (McMahon CM, Cancer Discov 2019). Previously, we demonstrated that TP‐0930, a multi‐kinase inhibitor, targets FLT3‐ITD and resistance‐conferring tyrosine kinase domain mutations in preclinical models. Here, we present the activity of TP‐0903 in RAS pathway mutant models of AML.KiNativ assay was performed in OCI‐AML3 cells (NRAS Q61L) treated with TP‐0903 (100 nM, 2h). Inhibition of cell viability was evaluated in AML cell lines (MTT) and primary samples (CellTiter Glo). Inhibition of AURKA/B, AKT, ERK, MCL‐1, and BCL‐2 were determined by western blot. Apoptosis, cell cycle, and differentiation assays were performed in OCI‐AML3 using annexin V, DAPI and CD11b‐APC staining. In vivo activity of TP‐0903 was evaluated in a systemic OCI‐AML3‐Luc+ xenograft model; 5e5 luciferase expressing cells were tail vein injected to NSG mice and TP‐0903 50 mg/kg or vehicle was given orally once dailyx5/week. Progression of leukemia was monitored via bioluminescence imaging and Kaplan Meier was used for survival analysis.In Kinativ assay, inhibition of native kinases were similar to those observed in a previous binding assay. We observed that TP‐0903 inhibited ACK1 (activated CDC42 kinase 1 or TNK2) and GCK (germinal center kinase or MAP4K2) by 87 and 46%, respectively. Inhibition of ACK1 (Kd = 7.3 nM, IC50 = 30.7 nM) and GCK (Kd = 1.8 nM, IC50 = 2.5 nM) was confirmed in binding and kinase assays. In RAS mutant AML cell lines (OCI‐AML3, THP1, HL60, and U937), TP‐0903 inhibited viability with IC50 values of 26 – 99 nM. In OCI‐AML3, TP‐0903 at 100 nM inhibited pAURKA/B, pAKT and MCL‐1; caused a G2‐M arrest and polyploidy (20 nM, 24h); and induced apoptosis (20–50 nM, 72h). TP‐0903, compared to gilteritinib, was more potent in inhibiting 4 ex vivo human primary AML samples with N/KRAS mutations co‐occurring with wt FLT3 or FLT3‐ITD, along with other mutations (IC50 range, 21 – 60 vs 260 – 1370 nM). In a 14‐day CFU assay, TP‐0903 inhibited colony formation of a FLT3‐ITD and NRAS mutant primary sample compared to DMSO (mean, 65 vs 188 CFUs). In an OCI‐AML3‐Luc+ xenograft study, TP‐0903 suppressed the outgrowth of leukemia at day 21 (P&lt;0.0001) and prolonged median survival by 9 days compared to veh‐treated mice (P&lt;0.0001). At study endpoint, spleen weight was higher in veh‐treated mice than in TP‐0903‐treated mice (mean ± SD, 333 ± 23 vs 181 ± 21 mg, P=0.0029).TP‐0903, a novel multi‐kinase inhibitor, shows promising in vitro and in vivo activity in RAS mutant AML. In a previous report (Nonami A, Blood 2015), an integrated approach involving cell‐based pharmacologic screening combined with KiNativ, gene expression profiling and mechanism studies, identified 2 kinase signaling pathways (ACK1/AKT and GCK) that synergistically contributed to the growth of NRAS mutant leukemia cells, including OCI‐AML3. Based on our data, TP‐0903 may exert its activity in RAS mutant AML via inhibiting ACK1 and GCK.

Wild-type FLT3 and FLT3 ITD exhibit similar ligand-induced internalization characteristics.

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.

Incidence, Risk Factors, Treatment and Long Term Outcome of Refractory and First Relapse AML Patients in the OSHO Studies

Incidence, Risk Factors, Treatment and Long Term Outcome of Refractory and First Relapse AML Patients in the OSHO Studies

Abstract 2990: Individualized functional approach to tailoring acute myeloid leukemia therapy

Abstract The major focus of drug development in AML has shifted to targeted agents because, despite initial sensitivity to conventional chemotherapy, durable remissions are limited. The clinical implementation of effective targeted therapies still lags due to the lack of predictive biomarkers. To make the best use of existing therapy and to identify new targeted therapies we adopted a broadly applicable functional approach to precision medicine called “dynamic BH3 profiling” (DBP). DBP measures early death signaling induced by short-term drug exposure. Increased cell death signaling is reflected by increased mitochondrial sensitivity (priming) to standardized BH3 peptides mimicking pro-apoptotic proteins. To develop a personalized therapeutic strategy for AML using DBP, we utilized 17 patient-derived xenografts (PDX) established from de novo, primary refractory or relapsed (R/R) patients. Human myeloblasts from xenotransplanted mice were exposed to 30 targeted and 3 standard of care drugs to determine mitochondrial responses via DBP. Unsupervised clustering of ex vivo DBP responses segregated PDXs into two major clusters, where treatment naïve PDXs clustered distinctly from R/R PDXs. Most drugs induced priming in only selective PDXs, including kinase inhibitors, epigenetic modifiers, and a SMAC mimetic. In contrast, BH3 mimetics and CDK9 inhibitors showed activity across a majority of PDXs. Next, we validated the ability of DBP to predict in vivo responses of single-agent birinapant (SMAC mimetic), JQ-1 (BRD-4 inhibitor), quizartinib (FLT-3 inhibitor), and venetoclax (BCL-2 inhibitor) across 6 AML PDX models, prioritized based on their greatest range of priming responses. The models that showed increased ex vivo priming via DBP also showed greatest in vivo responses, indicating that DBP can rank PDXs according to their drug sensitivities (AUC of ROC 0.87, p&amp;lt;0.005). By comparing the predictive power of DBP to other precision medicine tools such as genomics, we found that DBP was able to accurately predict quizartinib activity in PDXs with WT FLT-3, which are categorized as unresponsive based on genomics. To test the applicability of DBP in assigning therapies in the relapsed setting, we induced resistance to PDX models to single agents via long-term in vivo selection. Myeloblasts of relapsing clone showed reduced baseline mitochondrial priming and loss of sensitivity to most of the agents. This suggested that acquired resistance to single agents selects for apoptosis-refractory clones which then drive a pan-drug resistant phenotype. Finally, we applied this approach to humans, showing that the pretreatment mitochondrial apoptotic priming determined by DBP identifies responders to lenalidomide plus MEC combination therapy in the phase 1 trial of R/R AML patients. In summary, our results suggest that mitochondria-based measurements may serve as a precision medicine tool to guide therapy for a heterogeneous population. Citation Format: Shruti Bhatt, Binyam Yilma, Elyse Olesinski, Holly Zhu, Mark Murakami, Vineeth Kumar Murali, Sophia Adamia, David M. Weinstock, Jacqueline S. Garcia, Anthony Letai. Individualized functional approach to tailoring acute myeloid leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2990.

Abstract 1323: CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients

Abstract CG-806 is a pan-FLT3 / pan-BTK inhibitor that is more potent (IC50 = 0.08 µM, n=265, p &amp;lt; 0.001) than other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib, crenolanib and gilteritinib against acute myeloid leukemia (AML) primary patient samples containing wild-type or mutated FLT3. CG-806 has significant activity against AML cell lines with or without FLT3 internal tandem duplication (ITD) / tyrosine kinase domain (TKD) mutations and in mouse AML xenograft models. Oral CG-806 has a desirable safety profile in the pre-IND studies of rodent and dog 28-day GLP toxicology, rodent respiratory and central nervous system safety, and the bacterial reverse mutation assay. The current study explored the relationship between genetic abnormalities in bone marrow and peripheral mononuclear cells isolated from AML patients and sensitivity to CG-806 using an ex vivo cytotoxicity assay. To correlate CG-806 sensitivity with clinical status, gene abnormalities and expression levels, whole exome sequencing (n=118) and RNA sequencing (n=111) were performed. CG-806 was equally potent against cells from patients in the adverse, intermediate and favorable risk groups (2017 ELN risk stratification), and cells from patients with relapsed or transformed AML (WHO classification) were as sensitive as those from patients with de novo AML. CG-806 had equivalent potency in cases of TP53 WT and TP53 mutations, whereas cases with TP53 mutations were resistant (FDR-corrected p&amp;lt;0.1) to most other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib and crenolanib. CG-806 had similar potency in cases with ASXL1 or SRSF2 mutations compared to WT, whereas sunitinib and crenolanib appeared resistant to ASXL1 and SRSF2 mutations, respectively. As expected, patient samples with FLT3-ITD mutation were more sensitive to CG-806 as compared to FLT3 WT (FDR-corrected p&amp;lt;0.01); in addition, cases with high ITD allelic ratio, including concurrent mutations with NPM1 and DNMT3A, had greater sensitivity than cases with low allelic ratio. Most unexpectedly, all 6 specimens containing IDH1 R132 mutations demonstrated significantly greater sensitivity to CG-806 relative to WT (FDR-corrected p&amp;lt;0.01), yet there was no increased sensitivity of IDH-2 mutant cells to CG-806. In conclusion, CG-806 demonstrated potency in primary AML patient samples across all AML subgroups including relapsed/refractory/transformed AML and those with genetic abnormalities related to poor prognosis. While patient samples with FLT3-ITD mutations were expected to have greater sensitivity to CG-806, the most surprising correlation was the sensitivity of patient samples with IDH1 R132 mutations. These features of CG-806 warrant investigation in the clinical setting. Citation Format: Hongying Zhang, Andrea Local, Jeffrey W. Tyner, Stephen E. Kurtz, Beth Wilmot, Shannon Mcweeney, Brian J. Druker, Stephen B. Howell, William G. Rice. CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1323.

PF204 TARP AS IMMUNOTHERAPEUTIC TARGET IN AML EXPRESSED IN THE LSC COMPARTMENT

Background:Acute myeloid leukemia (AML) accounts for 80% of adult and 20% of pediatric leukemias. Despite frequent initial remission, patients exhibit a high relapse risk, probably due to therapy‐resistant leukemic stem cells (LSCs). Particularly the prognosis of FLT3‐ITD harboring patients remains extremely poor.Aims:To identify LSC‐specific targets and develop immunotherapeutic strategies for LSC elimination while assuring salvage of normal hematopoietic stem cells (HSCs).Methods:The GSE 17054 micro‐array dataset was used for transcriptome analysis of CD34+CD38‐ populations sorted from de novo adult AML (LSCs, n = 9) and healthy adults (HSCs, n = 4). We next evaluated transcript expression in CD34+CD38‐ and CD34+CD38+ populations sorted from de novo pediatric AML (pedAML) and healthy controls. An 8x60K human gene expression micro‐array was performed in four pediatric AML (pedAML) (2 FLT3 WT, 2 FLT3‐ITD) and 3 cord blood samples. In addition, qPCR was performed in 13 pedAML, 17 adult AML, and 15 controls (cord blood, normal bone marrow and mobilized blood stem cells). Finally, cell lines of various origin, including 9 AML cell lines, were examined. Protein expression was evaluated by Western blotting and confocal microscopy. Viral transduction was used to generate T‐cell receptor (TCR)‐engineered cytotoxic T‐cells (CTLs), next to HLA‐A∗0201 expressing, TARP overexpression and TARP knockdown AML cell lines. Killing by TCR‐engineered CTLs was evaluated in vitro by cytokine assays, flow cytometry‐ and bioluminescence‐based killing assays.Results:Re‐analysis of the GSE 17054 dataset revealed that the TCRγ chain alternate reading frame protein (TARP) was the most differentially expressed gene between LSC and HSC. Until now, TARP is known as a prostate‐specific marker, also expressed in breast adenocarcinoma. Subsequently, micro‐array and qPCR analysis showed aberrantly high TARP expression within pedAML and adult AML leukemic blasts and LSCs, while absent in normal counterparts. Noteworthy, all FLT3‐ITD harboring pedAML patients (n = 8) showed high TARP expression (P &lt; 0.01), whereas this association was not significant for FLT3‐ITD adult AML (n = 9). No TARP transcripts were identified in B‐ALL, CML nor B‐cell lines, while expression in AML cell lines was significantly higher (P &lt; 0.001). Aberrant TARP expression in cell lines and primary patient samples was confirmed at the protein level and confocal microscopy further corroborated these data. Importantly, cell lines and primary patient samples co‐expressing TARP and HLA‐A∗0201 were efficiently killed by TARP‐TCR engineered CTLs, providing proof‐of‐concept that TARP qualifies as a physiologically relevant target for T‐cell therapy in AML.Summary/Conclusion:We showed that TARP is highly expressed in AML leukemic cells, including LSCs, while absent in normal counterparts. High TARP expression is significantly associated with FLT3‐ITD in pedAML, a marker for poor prognosis. TARP‐TCR engineered CTLs effectively killed TARP+ HLA‐A∗0201+ co‐expressing AML cell lines and primary leukemic cells in vitro. To the best of our knowledge, this is the first report indicating that TARP‐TCR engineered CTLs hold great promise for immunotherapeutic T‐cell therapy in AML.

Treatment patterns and healthcare resource utilization in patients with FLT3-mutated and wild-type acute myeloid leukemia: A medical chart study.

ObjectivesTo assess real‐world treatment patterns and healthcare resource utilization (HRU) among patients with FLT3–mutated (FLT3 mut) and FLT3–wild‐type (FLT3 wt) acute myeloid leukemia (AML).MethodsData were abstracted from medical charts of patients with AML from 10 countries. Patients were grouped based on their FLT3 mutation status, age (18‐64 or ≥65), and whether they were newly diagnosed (ND) or relapsed/refractory (R/R).ResultsCharts of 1027 AML patients were included (183 FLT3 mut 18‐64 ND; 136 FLT3 mut ≥65 ND; 181 FLT3 mut R/R; 186 FLT3 wt 18‐64 ND; 159 FLT3 wt ≥65 ND; 182 FLT3 wt R/R). Substantial heterogeneity was observed in treatment patterns for AML. Among ND patients 18‐64, the most common initial treatment was standard‐to‐intermediate dose cytarabine‐based therapies (43.2% for FLT3 mut and 55.9% for FLT3 wt); among ND patients ≥65, the most common initial treatment was hypomethylating agent‐based therapies (36.0% and 47.2%). Among R/R patients, the most common initial treatment after R/R was best supportive care only (39.8% and 24.7%). HRU was substantial across cohorts during both event‐free and post‐event periods.ConclusionsTreatment patterns of AML were heterogeneous and FLT3 mut AML was treated more aggressively than FLT3 wt disease. HRU was substantial for all cohorts, particularly after relapse or treatment failure.