Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.

The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, our understanding of the relationships between LSC and pre-leukemic cells is still incomplete. In particular, it is not known whether "niche-anchoring" of pre-leukemic cell affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C expressed by haematopoietic stem cells (HSC) and LSC in an inducible iMLL-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long term to short term-HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSC isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to Activation Protein-1 (AP-1) and TNF-/NFB pathways. Human orthologs of dysregulated genes allowed to identify a score based on AP-1/TNF-a gene expression that was distinct and complementary from LSC-17 score. Sub-stratification of AML patients with LSC-17 and AP-1/TNF-genes signature defined four groups with median survival ranging from below one year to a median not reached after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF- gene signature as a proxy of LSC anchoring in specific bone marrow niches which improves the prognosis value of the LSC-17 score. NCT02320656.

Outcomes of acute myeloid leukemia patients who responded to venetoclax and azacitidine and stopped treatment.

Venetoclax-azacitidine is the standard of treatment for unfit acute myeloid leukemia patients. In the VIALE-A study, treatment was given until progression but there are no data on its optimal duration for responding patients who do not tolerate indefinite therapy. We retrospectively analyzed the outcome of patients who discontinued venetoclax or venetoclax-azacitidine due to poor tolerance. Sixty-two newly diagnosed (ND) AML patients and 22 patients with morphological relapse or refractory AML were included. In the ND cohort (n = 62), 28 patients stopped venetoclax and azacitidine and 34 patients continued azacitidine monotherapy. With a median follow-up of 23 months (IQR, 20-32), median overall survival and treatment-free survival were 44 (IQR, 16-NR) and 16 (IQR, 8-27) months, respectively. Patients who stopped both treatments and those who continued azacitidine monotherapy had the same outcomes. Negative minimal residual disease was associated with a 2-year treatment-free survival of 80%. In the RR cohort (n = 22), median overall survival and treatment-free survival were 19 (IQR, 17-31) and 10 (IQR, 5-NR) months, respectively. Prior number of venetoclax-azacitidine cycles and IDH mutations were associated with increased overall survival. The only factor significantly impacting treatment-free survival was the number of prior cycles. This study suggests that patients who discontinued treatment in remission have favorable outcomes supporting the rationale for prospective controlled trials.

Prophylactic versus restrictive platelet transfusion strategy in patients with haematological malignancies in the ICU setting, a propensity-score analysis.

PURPOSE: Prophylactic platelet transfusions (PT) aim to reduce bleeding. We assessed whether restrictive PT compared to prophylactic strategy could apply in ICU. MATERIAL AND METHODS: We conducted a retrospective monocentric study including patients >18 yo with haematological malignancy admitted to the ICU with thrombocytopenia <20 G/L between 2018 and 2021. Patients were classified in 2 groups according transfusion strategy applied during the first 3 days: prophylactic or restrictive transfusion. RESULTS: 180 patients were included, 87 and 93 in the restrictive and prophylactic groups respectively. After propensity-score analysis, 2 groups of 54 matched patients were analyzed. Restrictive strategy led to a significant reduction in PT with incidence rate for 100-ICU-patients-days of 34.9 and 49.9, incidence rate ratio = 0.699 [0.5-0.9], p = 0.006, representing a 31% decrease. Decreased PT persisted until day 28 with platelet concentrates transfusions-free days at day 28 of 21 [13-25] and 16.5 [10.2-21] in the 2 groups (p = 0.04). Restrictive strategy did not result in higher grade ≥ 2 bleeding. Transfusion efficiency was low with similar number of days with platelet <10 or < 20 G/L regardless of strategy. Platelet transfusion strategy was not associated with 28-day mortality. Platelet nadir <5G/L was associated with day-28 mortality with HR = 1.882 [1.011-3.055], p = 0.046. CONCLUSION: A restrictive PT strategy appears feasible in the ICU.

Poor prognosis of SRSF2 gene mutations in patients treated with VEN-AZA for newly diagnosed acute myeloid leukemia.

Mutations in spliceosome genes (SRSF2, SF3B1, U2AF1, ZRSR2) correlate with inferior outcomes in patients treated with intensive chemotherapy for Acute Myeloid Leukemia. However, their prognostic impact in patients treated with less intensive protocols is not well known. This study aimed to evaluate the impact of Spliceosome mutations in patients treated with Venetoclax and Azacitidine for newly diagnosed AML. 117 patients treated in 3 different hospitals were included in the analysis. 34 harbored a mutation in at least one of the spliceosome genes (splice-mut cohort). K/NRAS mutations were more frequent in the splice-mut cohort (47% vs 19%, p=0.0022). Response rates did not differ between splice-mut and splice-wt cohorts. With a median follow-up of 15 months, splice mutations were associated with a lower 18-month LFS (p=0.0045). When analyzing splice mutations separately, we found SRSF2 mutations to be associated with poorer outcomes (p=0.034 and p=0.037 for OS and LFS respectively). This negative prognostic impact remained true in our multivariate analysis. We believe this finding should warrant further studies aimed at overcoming this negative impact.

Venetoclax Resistance in Acute Myeloid Leukemia.

Venetoclax is a BH3-mimetics agent interacting with the anti-apoptotic protein BCL2, facilitating cytochrome c release from mitochondria, subsequent caspases activation, and cell death. Venetoclax combined with azacitidine (VEN-AZA) has become a new standard treatment for AML patients unfit for intensive chemotherapy. In the phase III VIALE-A study, VEN-AZA showed a 65% overall response rate and 14.7 months overall survival in comparison with 22% and 8 months in the azacitidine monotherapy control arm. Despite these promising results, relapses and primary resistance to venetoclax are frequent and remain an unmet clinical need. Clinical and preclinical studies have been conducted to identify factors driving resistance. Among them, the most documented are molecular alterations including IDH, FLT3, TP53, and the newly described BAX mutations. Several non-genetic factors are also described such as metabolic plasticity, changes in anti-apoptotic protein expression, and dependencies, as well as monocytic differentiation status. Strategies to overcome venetoclax resistance are being developed in clinical trials, including triplet therapies with targeted agents targeting IDH, FLT3, as well as the recently developed menin inhibitors or immunotherapies such as antibody-drug conjugated or monoclonal antibodies. A better understanding of the molecular factors driving venetoclax resistance by single-cell analyses will help the discovery of new therapeutic strategies in the future.

Peripheral blood haploidentical hematopoietic cell transplantation for patients aged 70 years and over with acute myeloid leukemia or high-risk myelodysplastic syndrome.

Haploidentical stem cell transplantation (Haplo-SCT) using non-myeloablative conditioning regimen (NMAC) has extended the feasibility of allogeneic transplantation, notably in older patients. However, there is few data specifically focusing on patients aged 70 years and over with AML and MDS. Thus the benefit of transplantation in this population is still debated. Here we report our single center experience of peripheral blood Haplo-SCT with NMAC and post-transplantation cyclophosphamide in AML and MDS patients aged 70 years and over. We analyzed 50 patients (27 AML, 23 MDS) with a median age of 72 years (70-77), 12/50 (24%) with active disease at Haplo-SCT. Cumulative incidence of grade 3-4 acute and moderate or severe chronic GVHD were 6% and 25%, respectively. Non-relapse mortality (NRM) at day +100 was 0%. NRM, relapse, PFS and OS at 3 years were 16%, 18%, 66%, and 69%, respectively. Among patients who were disease free at 2 years post Haplo-SCT, 88% are living without immunosuppressive treatment. Peripheral blood Haplo-SCT is feasible in selected AML/MDS patients over 70 years, without any early NRM. It produces long-term disease control and survival. Thus, age by itself should not be considered as a formal barrier to Haplo-SCT.

CPX-351 in higher risk myelodysplastic syndrome and chronic myelomonocytic leukaemia: a multicentre, single-arm, phase 2 study.

BACKGROUND: CPX-351, an encapsulated form of cytarabine and daunorubicin, has shown greater efficacy than the classic 3 + 7 treatment administration in secondary acute myeloid leukaemia. Given that higher-risk myelodysplastic syndrome and chronic myelomonocytic leukaemia share similarities with secondary acute myeloid leukaemia, we aimed to investigate the safety and efficacy of CPX-351 in this context. METHODS: This investigator-initiated two-cohort phase 2 trial was conducted by the Groupe Francophone des Myélodysplasies, with 12 participating centres in France. It comprised cohort A (reported here and completed), which included patients in first-line treatment, and cohort B, which was stopped for lack of inclusion (ie, not enough patients met the inclusion criteria), for patients with hypomethylating agent failure that is not reported here. Cohort A enrolled patients with newly diagnosed higher-risk myelodysplastic syndrome or chronic myelomonocytic leukaemia (aged 18-70 years old) with an Eastern Cooperative Oncology Group performance status of 0-1. Intravenous CPX-351 (100 mg/m2 cytarabine and 44 mg/m2 daunorubicin) was given on days 1, 3, and 5, with a second induction cycle given (same daily dose on days 1 and 3) if at least a partial response was not reached. Patients who responded could receive up to four monthly consolidation cycles (same daily dose on day 1) or allogeneic haematopoietic stem-cell transplantation (HSCT). Overall response rate after one or two induction courses according to European LeukemiaNet 2017 acute myeloid leukaemia was the primary endpoint after CPX-351 induction, whether patients received one or two induction cycles. Safety was assessed in all patients enrolled (in cohort A). This trial is registered with ClinicalTrials.gov, NCT04273802. FINDINGS: Between April 29, 2020, and Feb 10, 2021, 21 (68%) male and ten (32%) female patients were enrolled. 27 (87%) of 31 patients responded (95% CI 70-96). 16 (52%) of the 31 patients received at least one consolidation cycle. 30 (97%) of the 31 patients included were initially considered eligible for allogeneic HSCT and 29 (94%) of the 31 patients had the procedure. Median follow-up was 16·1 months (IQR 8·3-18·1). The most common grade 3-4 adverse events were pulmonary (eight [26%] of 31 patients) and cardiovascular (six [19%] of 31 patients). There were 14 serious adverse events (mainly hospitalisation for infection [n=5] and only one was treatment-related) and no treatment-related death. INTERPRETATION: CPX-351 appears to be active and safe in patients with higher-risk myelodysplastic syndrome and chronic myelomonocytic leukaemia, allowing bridging to allogenic HSCT in most patients. FUNDING: Jazz Pharmaceuticals.

Erythroleukemia: Classification.

Acute erythroid leukemia (AEL) is a rare (2%-5%) form of acute myeloid leukemia (AML). Molecular alterations found in AEL resemble those of other AMLs. We report a classification of AELs in three major classes, with different prognosis and some specific features such as a tendency to mutual exclusion of mutations in epigenetic regulators and signaling genes.

FMS-Like Tyrosine Kinase 3 Inhibitors in the Treatment of Acute Myeloid Leukemia: An Update on the Emerging Evidence and Safety Profile.

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.

Gilteritinib activity in refractory or relapsed FLT3-mutated acute myeloid leukemia patients previously treated by intensive chemotherapy and midostaurin: a study from the French AML Intergroup ALFA/FILO.

The real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients. Among them, 140 received gilteritinib as single agent (cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (cohort C). The main differences in patient characteristics in this study compared to the ADMIRAL trial were ECOG ≥ 2 (83.6% vs. 16.6%), FLT3-TKD mutation (21.0% vs. 8.5%), primary induction failure (15.0% vs. 40.0%) and line of treatment (beyond 2nd in 37.1% vs. 0.0%). The rates of composite complete remission, excluding those that occurred after hematopoietic stem cell transplantation (HSCT), were similar at respectively 25.4% and 27.5% in cohorts B and C. Median overall survival (OS) for these two groups was also similar at respectively 6.4 and 7.8 months. Multivariate analyses for prognostic factors associated with OS identified female gender (HR 1.61), adverse cytogenetic risk (HR 2.52), and allogenic HSCT after gilteritinib (HR 0.13). Although these patients were more heavily pretreated, these real-world data reproduce the results of ADMIRAL and provide new insights into the course of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment who can benefit from treatment in an outpatient setting.

Hepatic haemophagocytosis in haematology patients with hepatic dysfunction: prognostic impact and contribution of liver biopsy combined with the haemophagocytic syndrome diagnostic score (HScore).

Hepatic dysfunction (HD) is common in patients with haematological malignancies. Hepatic haemophagocytosis (HH) was detected in >50% of liver biopsies taken when HD remained unresolved after standard examination. We aimed to explore the contribution of liver biopsy in patients with both haematological malignancies and HD, describe the population of patients with HH, assess the prognostic impact of HH, and investigate haemophagocytic syndrome diagnostic score (HScore) utility in patients with HH. Between 2016 and 2019, 116 consecutive liver biopsies (76 transjugular, 40 percutaneous) were taken in 110 patients with haematological malignancy and HD (hyperbilirubinaemia, elevated transaminases, and/or cholestasis) and without a clear diagnosis. Liver biopsies were safe and diagnostically efficient. Predominant diagnoses included: HH (56%), graft-versus-host disease (55%), associated infections (24%), sinusoidal obstruction syndrome (15%), and tumoral infiltration (8%). Of patients, 35% were critically ill and 74% were allogeneic haematopoietic stem cell transplantation recipients, while 1-year overall survival (OS) was 35% with HH versus 58% without HH (p = 0.026). The 1-year OS was 24% with a HScore of ≥169 versus 50% with a HScore of <169 (p = 0.019). Liver biopsies are feasible in and contribute significantly to haematology patients with HD. HH occurred frequently and was associated with a poor prognosis. Combined with liver biopsy, the HScore may be helpful in refining haemophagocytic syndrome diagnosis.

Azacitidine Plus Venetoclax for the Treatment of Relapsed and Newly Diagnosed Acute Myeloid Leukemia Patients.

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.

Venetoclax in Acute Myeloid Leukemia: Molecular Basis, Evidences for Preclinical and Clinical Efficacy and Strategies to Target Resistance.

Venetoclax is a BH3-mimetics agent specifically interacting with the antiapoptotic protein BCL-2, facilitating cytochrome c release from mitochondria, subsequent caspases activation, and cell death. Utilization of venetoclax has profoundly changed the landscape of treatment for the poor-prognosis category of AML patients unfit for intensive chemotherapy. In the phase III VIALE-A study, Venetoclax, in combination with the hypomethylating agent azacitidine, showed a 65% overall response rate and 14.7-month overall survival, in comparison with 22% and 8 months in the control arm. These results led to the widespread use of venetoclax in this indication. Other combination regimens, consisting of low-intensity, intensive, or targeted therapies are currently under evaluation. Despite promising results, preventing relapses or resistance to venetoclax is still an unmet clinical need. Numerous studies have been conducted to identify and overcome venetoclax resistance in preclinical models or in clinical trials, including the inhibition of other antiapoptotic proteins, the induction of proapoptotic BH3-only proteins, and/or the targeting of the mitochondrial metabolism and machinery.

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome.

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.

Myelodysplastic Syndromes: How to Recognize Risk and Avoid Acute Myeloid Leukemia Transformation.

PURPOSE OF REVIEW: To understand how myelodysplastic syndromes (MDS) transform to AML and to describe how transformation can be predicted and prevented. RECENT FINDINGS: Recent genomic analyses have shown that MDS progression to AML is associated with clonal expansion and clonal evolution. Mutation profiles of MDS change during progression and new mutations in signaling genes and transcription factors emerge. AML transformation can be predicted by several parameters including International Prognostic Scoring System IPSS risk category and transfusion requirements. The prognostic relevance of the acquisition of some gene mutations (i.e., IDH1 and 2, CBL, FT3, RAS, NPM1, TP53, and ASXL1) has to be prospectively validated. The most effective preventive therapy for AML transformation is allogeneic stem cell transplantation. Hypomethylating agents have been associated with prolonged time to AML transformation even in patients who did not achieve an objective response. The recent progress in the understanding of the molecular events leading to transformation and the event of new effective therapies open new avenues for a better prediction and prevention of AML transformation in patients with MDS.

Thiotepa, Fludarabine, and Busulfan Conditioning Regimen before T Cell-Replete Haploidentical Transplantation with Post-Transplant Cyclophosphamide for Acute Myeloid Leukemia: A Bicentric Experience of 100 Patients.

Haploidentical stem cell transplantation (haplo-SCT) with post-transplant cyclophosphamide (PT-Cy) is an alternative treatment for acute myeloid leukemia (AML) patients who lack HLA-matched donors. Relapse after haplo-SCT remains a major concern, especially after nonmyeloablative conditioning regimens. Promising results were reported for TBF-based conditioning regimens (thiotepa, busulfan, and fludarabine) in patients transplanted from different categories of donors and for various disease types but not specifically in PT-Cy haplo-SCT for AML. Here we evaluate the outcome of 100 AML patients who received haplo-SCT with PT-Cy after TBF conditioning regimens (reduced-intensity conditioning, n = 77; myeloablative conditioning, n = 23) in 2 transplant programs. Cumulative incidences of grades III to IV acute and moderate or severe chronic graft-versus-host disease (GVHD) were 7% and 14%, respectively. NRM at 2 years was 28%, significantly influenced by disease status at haplo-SCT (first complete response [CR1] versus advanced AML: 16% versus 38%, P = .016) but not by conditioning intensity or age. The cumulative incidences of relapse at 2 years were 17% and 24% in CR1 and advanced AML, respectively (not significant). Progression-free survival, overall survival, and GVHD and relapse-free survival at 2 years were 67%, 71%, and 49% in CR1 patients, respectively, whereas comparative values in patients with advanced disease were 37%, 41%, and 32%. Our study suggests that TBF conditioning for PT-Cy haplo-SCT is safe and effective for AML patients in CR1. In patients with more advanced disease, the relatively low incidence of relapse seems counterbalanced by a high nonrelapse mortality, underlining the need for alternative strategies to decrease relapse risk, without increasing the intensity of conditioning regimen.

FLT3 inhibitors: clinical potential in acute myeloid leukemia.

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.

Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells.

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.

Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.