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The treatment of blast phase chronic myeloid leukemia (bpCML) remains a challenge due at least in part to drug resistance of leukemia stem cells (LSCs). Recent clinical evidence suggests that the BCL-2 inhibitor venetoclax in combination with ABL-targeting tyrosine kinase inhibitors (TKIs) can eradicate bpCML LSCs. In this report, we employed preclinical models of bpCML to investigate the efficacy and underlying mechanism of LSC-targeting with venetoclax/TKI combinations. Transcriptional analysis of LSCs exposed to venetoclax and dasatinib revealed upregulation of genes involved in lysosomal biology, in particular lysosomal acid lipase A (LIPA), a regulator of free fatty acids. Metabolomic analysis confirmed increased levels of free fatty acids in response to venetoclax/dasatinib. Pre-treatment of leukemia cells with bafilomycin, a specific lysosome inhibitor, or genetic perturbation of LIPA, resulted in increased sensitivity of leukemia cells toward venetoclax/dasatinib, implicating LIPA in treatment resistance. Importantly, venetoclax/dasatinib treatment does not affect normal stem cell function, suggestive of a leukemia-specific response. These results demonstrate that venetoclax/dasatinib is an LSCselective regimen in bpCML and that disrupting LIPA and fatty acid transport enhances venetoclax/dasatinib response in targeting LSCs, providing a rationale for exploring lysosomal disruption as an adjunct therapeutic strategy to prolong disease remission.

Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broadscale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital polymerase chain reaction (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.

Abstract Background 21q22 amplification is a rare cytogenetic aberration in acute myeloid leukemia (AML). So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized. Case presentation Here, we describe a case series of three AML patients with amplified 21q22 identified by fluorescence in situ hybridization using a RUNX1 probe. Two of these patients presented with therapy-related AML (t-AML) secondary to chemotherapy, while the third had de novo AML. There was one case each of FAB M0, M1 and M4. Morphologic evidence of dysplasia was identified in both t-AML cases. Phenotypic abnormalities of the myeloblasts were frequently observed. Extra copies of 21q22 were present on chromosome 21 and at least one other chromosome in two cases. Two showed a highly complex karyotype. Microarray analysis of 21q22 amplification in one case demonstrated alternating levels of high copy number gain split within the RUNX1 locus at 21q22. The same patient also had mutated TP53. Two patients died at 1.5 and 11 months post-treatment, while the third elected palliative care and died within 2 weeks. Conclusions Our results provide further evidence that 21q22 amplification in AML is associated with complex karyotypes, TP53 aberrations, and poor outcomes. Furthermore, we demonstrate that 21q22 amplification is not always intrachromosomally localized to chromosome 21 and could be a result of structural aberrations involving 21q22 and other chromosomes.

Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: i) higher doses of venetoclax are tolerable and more effective, and ii) azacitidine can be discontinued after deep remissions. Forty-two newly diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine+Venetoclax (HiDDAV) Study (clinicaltrials gov. Identifier: NCT03466294). Patients received one to three “induction” cycles of venetoclax 600 mg daily with azacitidine. Responders received MRD-positive or MRDnegative “maintenance” arms: azacitidine with 400 mg venetoclax or 400 mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400 mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital polymerase chain recation. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses >400 mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.

Abstract Diagnostic and molecular genetic testing are key in advancing the treatment of acute myeloid leukemia (AML), yet little is known about testing patterns outside of clinical trials, especially in older patients. We analyzed diagnostic and molecular testing patterns over time in 565 patients aged ≥ 55 years with newly diagnosed AML enrolled in the Connect® MDS/AML Disease Registry (NCT01688011) in the United States. Diagnostic data were recorded at enrolment and compared with published guidelines. The percentage of bone marrow blasts was reported for 82.1% of patients, and cellularity was the most commonly reported bone marrow morphological feature. Flow cytometry, karyotyping, molecular testing, and fluorescence in situ hybridization were performed in 98.8%, 95.4%, 75.9%, and 75.7% of patients, respectively. Molecular testing was done more frequently at academic than community/government sites (84.3% vs 70.2%; P

Abstract Background The phase 2 BRIGHT AML 1003 trial evaluated efficacy and safety of glasdegib + low-dose cytarabine (LDAC) in patients with acute myeloid leukemia ineligible for intensive chemotherapy. The multicenter, open-label study randomized patients to receive glasdegib + LDAC (n = 78) or LDAC alone (n = 38). The rate of complete remission (CR) was 19.2% in the glasdegib + LDAC arm versus 2.6% in the LDAC arm (P = 0.015). Methods This post hoc analysis determines whether the clinical benefits of glasdegib are restricted to patients who achieve CR, or if they extend to those who do not achieve CR. Results In patients who did not achieve CR, the addition of glasdegib to LDAC improved overall survival (OS) versus LDAC alone (hazard ratio = 0.63 [95% confidence interval, 0.41–0.98]; P = 0.0182; median OS, 5.0 vs 4.1 months). Additionally, more patients receiving glasdegib + LDAC achieved durable recovery of absolute neutrophil count (≥ 1000/μl, 45.6% vs 35.5%), hemoglobin (≥ 9 g/dl, 54.4% vs 38.7%), and platelets (≥ 100,000/μl, 29.8% vs 9.7%). Transfusion independence was achieved by 15.0% and 2.9% of patients receiving glasdegib + LDAC and LDAC alone, respectively. Conclusions Collectively, these data suggest that there are clinical benefits with glasdegib in the absence of CR. Trial registration ClinicalTrials.gov NCT01546038 (March 7, 2012)

Abstract: Venetoclax is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore activation of apoptosis in malignancies, the survival of which depends on dysregulation of this pathway. Preclinical data, using various model systems including cell lines and patient samples, suggested targeting BCL-2 could be a successful therapeutic strategy in patients with acute myeloid leukemia (AML). As predicted by this work, the use of venetoclax in the clinical setting has resulted in promising outcomes for patients with this disease. Although venetoclax showed limited activity as a single agent in the relapsed disease setting, recent studies have shown that when combined with a backbone therapy of a hypomethylating agent or low-dose cytarabine, high response rates with encouraging remission durations for older patients with newly diagnosed AML who were not candidates for intensive induction chemotherapy were observed. Furthermore, venetoclax-based therapies allowed for rapid responses and were able to effectively target the leukemia stem cell population. Here we review the preclinical data that supported the development of venetoclax in AML, as well as the results of the promising clinical trials.

Abstract: Venetoclax is approved for older untreated acute myeloid leukemia (AML) patients. Venetoclax was available prior to approval off-label. We assessed our single-institution off-label experience with venetoclax/azacitidine, comparing outcomes with a clinical trial cohort that administered this regimen at the same institution. Thirty-three untreated AML patients unfit or unwilling to receive induction chemotherapy and prescribed venetoclax/azacitidine off-trial were retrospectively analyzed and compared with 33 patients who received the same therapy on trial. Outcomes were compared, and comparisons were made to a theoretical scenario in which off-trial patients received induction. Digital droplet polymerase chain reaction evaluated measurable residual disease (MRD). Off-trial venetoclax was attainable in nearly all patients for whom this was desired. The complete remission (CR)/CR with incomplete blood count recovery rate was 63.3% for off-trial patients who received treatment and 84.9% for trial patients (P = .081). The median overall survival for off-trial patients who received treatment was 381 days (95% confidence interval [CI], 174, not reached) vs 880 days (95% CI, 384, not reached) for trial patients (P = .041). Prior exposure to hypomethylating agents was associated with worse outcomes. Response rates with venetoclax/azacitidine were not inferior to a theoretical scenario in which patients received induction, and early death rates were less than expected with induction. MRD negativity was achievable. Newly diagnosed AML patients treated in a “real-world” scenario with off-trial venetoclax/azacitidine had inferior outcomes compared with patients treated in the setting of a clinical trial. Additionally, this therapy may be as effective, and less toxic, when compared with induction chemotherapy.

Myelodysplastic syndrome (MDS) arises from mutations in hematopoietic stem cells (HSCs). Here, the authors demonstrate that HSCs in higher-risk MDS express the surface marker CD123 and are characterized by activation of protein synthesis machinery and increased oxidative phosphorylation.

Secreted proteins in the bone marrow microenvironment play critical roles in acute myeloid leukemia (AML). Through an ex vivo functional screen of 94 cytokines, we identified that the pro-inflammatory cytokine interleukin-1 (IL-1) elicited profound expansion of myeloid progenitors in ∼67% of AML patients while suppressing the growth of normal progenitors. Levels of IL-1β and IL-1 receptors were increased in AML patients, and silencing of the IL-1 receptor led to significant suppression of clonogenicity and in vivo disease progression. IL-1 promoted AML cell growth by enhancing p38MAPK phosphorylation and promoting secretion of various other growth factors and inflammatory cytokines. Treatment with p38MAPK inhibitors reversed these effects and recovered normal CD34+ cells from IL-1-mediated growth suppression. These results highlight the importance of ex vivo functional screening to identify common and actionable extrinsic pathways in genetically heterogeneous malignancies and provide impetus for clinical development of IL-1/IL1R1/p38MAPK pathway-targeted therapies in AML.

Summary: The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs. : The NADPH-dependent oxidase NOX2 is important for normal myeloid cell function. Adane et al. show that NOX2 is expressed in leukemic stem cells, where it regulates the balance of myeloid differentiation and self-renewal. Deficiency of NOX2 altered core metabolism, exacerbated inflammatory signaling, and limited in vivo disease development. Keywords: acute myeloid leukemia, leukemia stem cells, differentiation, self-renewal, glycolysis, fatty acid oxidation, NOX2, p22Phox, ROS, FOXC1, CEBPε, NF-κB

Therapy of acute myeloid leukemia in older persons is associated with poor outcomes because of intolerance to intensive therapy, resistant disease and co-morbidities. This multi-center, randomized, open-label, phase II trial compared safety and efficacy of three therapeutic strategies in patients 65 years or over with newly-diagnosed acute myeloid leukemia: 1) continuous high-dose lenalidomide (n=15); 2) sequential azacitidine and lenalidomide (n=39); and 3) azacitidine only (n=34). The efficacy end point was 1-year survival. Median age was 76 years (range 66–87 years). Thirteen subjects (15%) had prior myelodysplastic syndrome and 41 (47%) had adverse cytogenetics. One-year survival was 21% [95% confidence interval (CI): 0, 43%] with high-dose lenalidomide, 44% (95%CI: 28, 60%) with sequential azacitidine and lenalidomide, and 52% (95%CI: 35, 70%) with azacitidine only. Lenalidomide at a continuous high-dose schedule was poorly-tolerated resulting in a high rate of early therapy discontinuations. Hazard of death in the first four months was greatest in subjects receiving continuous high-dose lenalidomide; hazards of death thereafter were similar. These data do not favor use of continuous high-dose lenalidomide or sequential azacitidine and lenalidomide over the conventional dose and schedule of azacitidine only in patients aged 65 years or over with newly-diagnosed acute myeloid leukemia. (clinicaltrials.gov identifier: 01358734).

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1− subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1− cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1− leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1− leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.

Despite an increasingly rich understanding of its pathogenesis, acute myeloid leukemia remains a disease with poor outcomes, overwhelmingly due to disease relapse. In recent years, work to characterize the leukemia stem cell population, the disease compartment most difficult to eliminate with conventional therapy and most responsible for relapse, has been undertaken. This, in conjunction with advances in drug development that have allowed for increasingly targeted therapies to be engineered, raises the hope that we are entering an era in which the leukemia stem cell population can be eliminated, resulting in therapeutic cures for acute myeloid leukemia patients. For these therapies to become available, they must be tested in the setting of clinical trials. A long-established clinical trials infrastructure has been employed to shepherd new therapies from proof-of-concept to approval. However, due to the unique features of leukemia stem cells, drugs that are designed to specifically eliminate this population may not be adequately tested when applied to this model. Therefore, in this review article, we seek to identify the relevant features of acute myeloid leukemia stem cells for clinical trialists, discuss potential strategies to target leukemia stem cells, and propose a set of guidelines outlining the necessary elements of clinical trials to allow for the successful testing of stem cell-directed therapies.

There are limited treatment options for older patients with acute myeloid leukemia and prognosis of these patients remains poor, thereby warranting development of novel therapies. We evaluated the efficacy and safety of azacitidine in combination with lenalidomide as front-line therapy for older patients with acute myeloid leukemia. Patients ≥60 years of age with untreated acute myeloid leukemia received azacitidine 75 mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 days starting on day 8 of each cycle every 6 weeks. Patients received continued therapy until disease progression, unacceptable toxicity, or completion of 12 cycles. Forty-two patients (median age, 74 years) were enrolled with equal distribution according to European LeukemiaNet risk. The overall response rate was 40% (rate of complete remission with or without complete recovery of blood counts = 28%). The median time to complete remission with or without complete recovery of blood counts was 12 weeks, and duration of this status was 28 weeks (range, 4 - >104 weeks). Therapy-related acute myeloid leukemia and a high score on the Hematopoietic Cell Transplantation Comorbidity Index were negative predictors of response. Early death was noted in 17% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosuppression. In conclusion, a sequential combination of azacitidine plus lenalidomide has clinical activity in older patients with acute myeloid leukemia, and further studies of this combination are underway. This study is registered at www.clinicaltrials.gov as # NCT00890929.