Key Points• Bortezomib induces the degradation of FLT3-ITD through an autophagydependent mechanism that contributes to cell death.• This finding provides a mechanism-based rationale for the study of proteasome inhibitors in FLT3-ITD-mutant acute myeloid leukemia.Internal tandem duplication of the Fms-like tyrosine kinase-3 receptor (FLT3) internal tandem duplication (ITD) is found in 30% of acute myeloid leukemia (AML) and is associated with a poor outcome. In addition to tyrosine kinase inhibitors, therapeutic strategies that modulate the expression of FLT3-ITD are also promising. We show that AML samples bearing FLT3-ITD mutations are more sensitive to proteasome inhibitors than wild-type samples and this sensitivity is strongly correlated with a higher FLT3-ITD allelic burden. Using pharmacologic inhibitors of autophagy, specific downregulation of key autophagy proteins including Vps34, autophagy gene (Atg)5, Atg12, Atg13, biochemical, and microscopy studies, we demonstrated that proteasome inhibitors induced cytotoxic autophagy in AML cells. FLT3-ITD molecules were detectable within autophagosomes after bortezomib treatment indicating that autophagy induction was responsible for the early degradation of FLT3-ITD, which preceded the inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), PI3K/AKT, and STAT5 pathways, and subsequent activation of cell death. Moreover, proteasome inhibitors overcome resistance to quizartinib induced by mutations in the kinase domain of FLT3, suggesting that these compounds may prevent the emergence of mutant clones arising from tyrosine kinase inhibitor treatments. In xenograft mice models, bortezomib stimulated the conversion of LC3-I to LC3-II, indicating induction of autophagy in vivo, downregulated FLT3-ITD protein expression and improved overall survival. Therefore, selecting patients according to FLT3-ITD mutations could be a new way to detect a significant clinical activity of proteasome inhibitors in AML patients. (Blood. 2016;127(7):882-892)

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy that is cured in as few as 15%–40% of cases. Tremendous improvements in AML prognostication arose from a comprehensive analysis of leukemia cell genomes. Among normal karyotype AML cases, mutations in the FLT3 gene are the ones most commonly detected as having a deleterious prognostic impact. FLT3 is a transmembrane tyrosine kinase receptor, and alterations of the FLT3 gene such as internal tandem duplications (FLT3-ITD) deregulate FLT3 downstream signaling pathways in favor of increased cell proliferation and survival. FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in FLT3-ITD AML, and clinical trials are ongoing with a variety of TKI either alone, combined with chemotherapy, or even as maintenance after allogenic stem cell transplantation. However, a wide range of molecular resistance mechanisms are activated upon TKI therapy, thus limiting their clinical impact. Massive research efforts are now ongoing to develop more efficient FLT3 TKI and/or new therapies targeting these resistance mechanisms to improve the prognosis of FLT3-ITD AML patients in the future.

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56−CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56−CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56−CD16+ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56−CD16+ NK cells. Overall, our data suggest that the accumulation of CD56−CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression.

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We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones.Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells.

Venetoclax (VEN) belongs the BH3-mimetic class that selectively targets BCL-2, activating apoptosis. The combination of VEN and azacitidine (AZA) has changed the paradigm of treatment of newly diagnosed (ND) acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy. There is scarce evidence for the use of VEN–AZA for relapsed or refractory (R/R) AML. We compared the outcome of 39 R/R AML and 38 ND AML patients treated between 01/20 and 12/21. The median age was 69 (22–86) and 73 (61–81) in the R/R and ND groups, respectively. Adverse cytogenetics were found in 36% of patients in the R/R group and 59% of patients in the ND group. Overall response rate was 37% in R/R AML, including 13% CR, 8% CRi, 3% PR and 13% MLFS, and 58% in the ND AML, including 32% CR, 13% CRi and 13% MLFS. Adverse cytogenetics was associated with treatment failure in the R/R group (Relative Risk = 0.13, p = 0.005). Median overall survival (OS) was 5.9 months in the R/R group and 9.4 months in the ND group. Median OS was 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group in the R/R group (p = 0.02). Median leukemia-free survival was not different between the two groups (9.4 months and 10.3 months), indicating that VEN–AZA can be an efficient salvage treatment for selected R/R AML patients. In conclusion, VEN–AZA is a promising treatment for ND AML and for selected R/R AML patients.

FMS-like tyrosine kinase 3 ( FLT3 ) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Approximately 30% of the adult cases harbor an internal tandem duplication ( FLT3 -ITD) and 5–10% a tyrosine kinase domain (TKD) amino acid substitution ( FLT3-TKD ). The treatment paradigm of AML patients harboring FLT3 mutations (30%) has been modified by the discovery of tyrosine kinase inhibitors. First- and second-generation inhibitors classify FLT3 inhibitors according to FLT3 specificity: first-generation FLT3 inhibitors include sorafenib and midostaurin and second-generation inhibitors are represented by quizartinib, gilteritinib and crenolanib, among others. Activity of these inhibitors depends on their mechanism of receptor binding (active vs inactive conformation) and efficacy against the FLT3-ITD and -TKD mutations (type 1 inhibitors are active both on FLT3 -ITD and TKD, whereas type 2 inhibitors are active only on FLT3 -ITD). The FLT3 inhibitors sorafenib, midostaurin, quizartinib and gilteritinib have been tested in monotherapy in several settings including refractory or relapsed AML (R/R AML), post-transplant maintenance as well as in combination with intensive chemotherapy (ICT) or non-intensity regimens. The results of published randomized studies support the use of sorafenib in a post-transplant setting (SORMAIN trial), midostaurin in combination with ICT based (RATIFY trial) and gilteritinib for R/R AML (ADMIRAL trial). Gilteritinib in combination with hypomethylating agent as well as quizartinib are not supported by solid randomized trial results for their use in FLT3-mutated AML patients.

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