Ex Vivo Venetoclax Sensitivity Predicts Clinical Response in Acute Myeloid Leukemia in the Prospective VenEx Trial.

The BCL2 inhibitor venetoclax has shown promise for treating acute myeloid leukemia (AML). However, identifying patients likely to respond remains a challenge, especially for those with relapsed/refractory (R/R) disease. We evaluated the utility of ex vivo venetoclax sensitivity testing to predict treatment responses to venetoclax-azacitidine in a prospective, multicenter, phase 2 trial conducted by the Finnish AML Group (VenEx, NCT04267081). The trial recruited 104 participants with previously untreated (n=48), R/R (n=39) or previously treated secondary AML (sAML) (n=17). The primary endpoint was complete remission or complete remission with incomplete hematologic recovery (CR/CRi) rate in ex vivo sensitive trial participants during the first three therapy cycles. The key secondary endpoints included the correlations between ex vivo drug sensitivity, responses, and survival. Venetoclax sensitivity was successfully assessed in 102/104 participants, with results available within a median of three days from sampling. In previously untreated AML, ex vivo sensitivity corresponded to an 85% (34/40) CR/CRi rate, with a median overall survival (OS) of 28.7 months, compared to 5.5 months for ex vivo resistant patients (p = 0.002). For R/R/sAML, ex vivo sensitivity resulted in a 62% CR/CRi rate (21/34) and median OS of 9.7 versus 3.3 months for ex vivo resistant patients (p < 0.001). In univariate and multivariate analysis, ex vivo venetoclax sensitivity was the strongest predictor for a favorable treatment response and survival. The VenEx trial demonstrates the feasibility of integrating ex vivo drug testing into clinical practice to identify AML patients, particularly in the R/R setting, who benefit from venetoclax.

Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia.

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Ex vivo venetoclax sensitivity testing predicts treatment response in acute myeloid leukemia.

The BCL-2 inhibitor venetoclax has revolutionized the treatment of acute myeloid leukemia (AML) in patients not benefiting from intensive chemotherapy. Nevertheless, treatment failure remains a challenge, and predictive markers are needed, particularly for relapsed or refractory AML. Ex vivo drug sensitivity testing may correlate with outcomes, but its prospective predictive value remains unexplored. Here we report the results of the first stage of the prospective phase II VenEx trial evaluating the utility and predictiveness of venetoclax sensitivity testing using different cell culture conditions and cell viability assays in patients receiving venetoclax-azacitidine. Participants with de novo AML ineligible for intensive chemotherapy, relapsed or refractory AML, or secondary AML were included. The primary endpoint was the treatment response in participants showing ex vivo sensitivity and the key secondary endpoints were the correlation of sensitivity with responses and survival. Venetoclax sensitivity testing was successful in 38/39 participants. Experimental conditions significantly influenced the predictive accuracy. Blast-specific venetoclax sensitivity measured in conditioned medium most accurately correlated with treatment outcomes; 88% of sensitive participants achieved a treatment response. The median survival was significantly longer for participants who were ex vivo-sensitive to venetoclax (14.6 months for venetoclax-sensitive patients vs. 3.5 for venetoclax-insensitive patients, P<0.001). This analysis illustrates the feasibility of integrating drug-response profiling into clinical practice and demonstrates excellent predictivity. This trial is registered with ClinicalTrials.gov identifier: NCT04267081.

Venetoclax triggers sublethal apoptotic signaling in venetoclax-resistant acute myeloid leukemia cells and induces vulnerability to PARP inhibition and azacitidine.

Venetoclax plus azacitidine treatment is clinically beneficial for elderly and unfit acute myeloid leukemia (AML) patients. However, the treatment is rarely curative, and relapse due to resistant disease eventually emerges. Since no current clinically feasible treatments are known to be effective at the state of acquired venetoclax resistance, this is becoming a major challenge in AML treatment. Studying venetoclax-resistant AML cell lines, we observed that venetoclax induced sublethal apoptotic signaling and DNA damage even though cell survival and growth were unaffected. This effect could be due to venetoclax inducing a sublethal degree of mitochondrial outer membrane permeabilization. Based on these results, we hypothesized that the sublethal apoptotic signaling induced by venetoclax could constitute a vulnerability in venetoclax-resistant AML cells. This was supported by screens with a broad collection of drugs, where we observed a synergistic effect between venetoclax and PARP inhibition in venetoclax-resistant cells. Additionally, the venetoclax-PARP inhibitor combination prevented the acquisition of venetoclax resistance in treatment naïve AML cell lines. Furthermore, the addition of azacitidine to the venetoclax-PARP inhibitor combination enhanced venetoclax induced DNA damage and exhibited exceptional sensitivity and long-term responses in the venetoclax-resistant AML cell lines and samples from AML patients that had clinically relapsed under venetoclax-azacitidine therapy. In conclusion, we mechanistically identify a new vulnerability in acquired venetoclax-resistant AML cells and identify PARP inhibition as a potential therapeutic approach to overcome acquired venetoclax resistance in AML.

Targeting CD33+ Acute Myeloid Leukemia with GLK-33, a Lintuzumab-Auristatin Conjugate with a Wide Therapeutic Window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.