Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL.

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

Evaluation of vecabrutinib as a model for noncovalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia.

Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

MCL-1 Inhibition Overcomes Anti-apoptotic Adaptation to Targeted Therapies in B-Cell Precursor Acute Lymphoblastic Leukemia.

Multiple targeted therapies are currently explored for pediatric and young adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. However, this new armamentarium of therapies faces an old problem: choosing the right treatment for each patient. The lack of predictive biomarkers is particularly worrying for pediatric patients since it impairs the implementation of new treatments in the clinic. In this study, we used the functional assay dynamic BH3 profiling (DBP) to evaluate two new treatments for BCP-ALL that could improve clinical outcome, especially for relapsed patients. We found that the MEK inhibitor trametinib and the multi-target tyrosine kinase inhibitor sunitinib exquisitely increased apoptotic priming in an NRAS-mutant and in a KMT2A-rearranged cell line presenting a high expression of FLT3, respectively. Following these observations, we sought to study potential adaptations to these treatments. Indeed, we identified with DBP anti-apoptotic changes in the BCL-2 family after treatment, particularly involving MCL-1 - a pro-survival strategy previously observed in adult cancers. To overcome this adaptation, we employed the BH3 mimetic S63845, a specific MCL-1 inhibitor, and evaluated its sequential addition to both kinase inhibitors to overcome resistance. We observed that the metronomic combination of both drugs with S63845 was synergistic and showed an increased efficacy compared to single agents. Similar observations were made in BCP-ALL KMT2A-rearranged PDX cells in response to sunitinib, showing an analogous DBP profile to the SEM cell line. These findings demonstrate that rational sequences of targeted agents with BH3 mimetics, now extensively explored in clinical trials, may improve treatment effectiveness by overcoming anti-apoptotic adaptations in BCP-ALL.

MicroRNA-497/195 is tumor suppressive and cooperates with CDKN2A/B in pediatric acute lymphoblastic leukemia.

We previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In a search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes. We found high expression of miR-497 and miR-195 (hereafter miR-497/195) in patient-derived xenograft samples with slow engraftment derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression, and we identified cyclin-dependent kinase 4 (CDK4)- and cyclin-D3 (CCND3)-mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195-mediated cell cycle regulation was underscored by finding (in an additional independent series of patient samples) that high expression of miR-497/195 together with a full sequence for CDKN2A and CDKN2B (CDKN2A/B) was associated with excellent outcome, whereas deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as a new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.

Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling.

Deregulated cell death pathways contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Intrinsic apoptosis signaling is regulated by different proapoptotic and antiapoptotic molecules: proapoptotic BCL-2 homology domain 3 (BH3) proteins activate prodeath molecules leading to cellular death, while antiapoptotic molecules including B-cell lymphoma 2 (BCL-2) prevent activation of prodeath proteins and counter-regulate apoptosis induction. Inhibition of these antiapoptotic regulators has become a promising strategy for anticancer treatment, but variable anticancer activities in different malignancies indicate the need for upfront identification of responsive patients. Here, we investigated the activity of the BCL-2 inhibitor venetoclax (VEN, ABT-199) in B-cell precursor acute lymphoblastic leukemia and found heterogeneous sensitivities in BCP-ALL cell lines and in a series of patient-derived primografts. To identify parameters of sensitivity and resistance, we evaluated genetic aberrations, gene-expression profiles, expression levels of apoptosis regulators, and functional apoptosis parameters analyzed by mitochondrial profiling using recombinant BH3-like peptides. Importantly, ex vivo VEN sensitivity was most accurately associated with functional BCL-2 dependence detected by BH3 profiling. Modeling clinical application of VEN in a preclinical trial in a set of individual ALL primografts, we identified that leukemia-free survival of VEN treated mice was precisely determined by functional BCL-2 dependence. Moreover, the predictive value of ex vivo measured functional BCL-2 dependence for preclinical in vivo VEN response was confirmed in an independent set of primograft ALL including T- and high risk-ALL. Thus, integrative analysis of the apoptosis signaling indicating mitochondrial addiction to BCL-2 accurately predicts antileukemia activity of VEN, robustly identifies VEN-responsive patients, and provides information for stratification and clinical guidance in future clinical applications of VEN in patients with ALL.

Tight regulation of FOXO1 is essential for maintenance of B-cell precursor acute lymphoblastic leukemia.

The FOXO1 transcription factor plays an essential role in the regulation of proliferation and survival programs at early stages of B-cell differentiation. Here, we show that tightly regulated FOXO1 activity is essential for maintenance of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Genetic and pharmacological inactivation of FOXO1 in BCP-ALL cell lines produced a strong antileukemic effect associated with CCND3 downregulation. Moreover, we demonstrated that CCND3 expression is critical for BCP-ALL survival and that overexpression of CCND3 protected BCP-ALL cell lines from growth arrest and apoptosis induced by FOXO1 inactivation. Most importantly, pharmacological inhibition of FOXO1 showed antileukemia activity on several primary, patient-derived, pediatric ALL xenografts with effective leukemia reduction in the hematopoietic, lymphoid, and central nervous system organ compartments, ultimately leading to prolonged survival without leukemia reoccurrence in a preclinical in vivo model of BCP-ALL. These results suggest that repression of FOXO1 might be a feasible approach for the treatment of BCP-ALL.

Leukemia reconstitution in vivo is driven by cells in early cell cycle and low metabolic state.

In contrast to well-established hierarchical concepts of tumor stem cells, leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia have not yet been phenotypically identified. Different subpopulations, as defined by surface markers, have shown equal abilities to reconstitute leukemia upon transplantation into immunodeficient mice. Using a non-obese diabetes/severe combined immunodeficiency human acute lymphoblastic leukemia mouse model and cell cycle analysis annotating cells to distinct cycle phases, we functionally characterized leukemia-initiating cells and found that cells in all stages of the cell cycle are able to reconstitute leukemia in vivo, with early cycling cells (G1blow population) exhibiting the highest leukemia-initiating potential. Interestingly, cells of the G2/M compartment, i.e. dividing cells, were less effective in leukemia reconstitution. Moreover, G1blow cells were more resistant to spontaneous or drug-induced cell death in vitro, were enriched for stem cell signatures and were less metabolically active, as determined by lower levels of reactive oxygen species, compared to G2/M stage cells. Our data provide new information on the biological properties of leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia and underline the concept of a stochastic model of leukemogenesis.

Central nervous system involvement in acute lymphoblastic leukemia is mediated by vascular endothelial growth factor.

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement is a major clinical concern. Despite nondetectable CNS leukemia in many cases, prophylactic CNS-directed conventional intrathecal chemotherapy is required for relapse-free survival, indicating subclinical CNS manifestation in most patients. However, CNS-directed therapy is associated with long-term sequelae, including neurocognitive deficits and secondary neoplasms. Therefore, molecular mechanisms and pathways mediating leukemia-cell entry into the CNS need to be understood to identify targets for prophylactic and therapeutic interventions and develop alternative CNS-directed treatment strategies. In this study, we analyzed leukemia-cell entry into the CNS using a primograft ALL mouse model. We found that primary ALL cells transplanted onto nonobese diabetic/severe combined immunodeficiency mice faithfully recapitulated clinical and pathological features of meningeal infiltration seen in patients with ALL. ALL cells that had entered the CNS and were infiltrating the meninges were characterized by high expression of vascular endothelial growth factor A (VEGF). Although cellular viability, growth, proliferation, and survival of ALL cells were found to be independent of VEGF, transendothelial migration through CNS microvascular endothelial cells was regulated by VEGF. The importance of VEGF produced by ALL cells in mediating leukemia-cell entry into the CNS and leptomeningeal infiltration was further demonstrated by specific reduction of CNS leukemia on in vivo VEGF capture by the anti-VEGF antibody bevacizumab. Thus, we identified a mechanism of ALL-cell entry into the CNS, which by targeting VEGF signaling may serve as a novel strategy to control CNS leukemia in patients, replacing conventional CNS-toxic treatment.