L-Asparaginase (L-ASNase) is an enzyme that hydrolyses the amino acid asparagine into aspartic acid and ammonia. Systemic administration of bacterial L-ASNase is successfully used to lower the bioavailability of this non-essential amino acid and to eradicate rapidly proliferating cancer cells with a high demand for exogenous asparagine. Currently, it is a cornerstone drug in the treatment of the most common pediatric cancer, acute lymphoblastic leukemia (ALL). Since these lymphoblasts lack the expression of asparagine synthetase (ASNS), these cells depend on the uptake of extracellular asparagine for survival. Interestingly, recent reports have illustrated that L-ASNase may also have clinical potential for the treatment of other aggressive subtypes of hematological or solid cancers. However, immunogenic and other severe adverse side effects limit optimal clinical use and often lead to treatment discontinuation. The design of optimized and novel L-ASNase formulations provides opportunities to overcome these limitations. In addition, identification of multiple L-ASNase resistance mechanisms, including ASNS promoter reactivation and desensitization, has fueled research into promising novel drug combinations to overcome chemoresistance. In this review, we discuss recent insights into L-ASNase adverse effects, resistance both in hematological and solid tumors, and how novel L-ASNase variants and drug combinations can expand its clinical applicability.

B-cell lymphoma 2 (BCL-2) has recently emerged as a therapeutic target for early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), a high-risk subtype of human T-cell ALL. The major clinical challenge with targeted therapeutics, such as the BCL-2 inhibitor ABT-199, is the development of acquired resistance. We assessed the in vivo response of luciferase-positive LOUCY cells to ABT-199 monotherapy and observed specific residual disease in the splenic microenvironment. Of note, these results were confirmed by using a primary ETP-ALL patient-derived xenograft. Splenomegaly has previously been associated with poor prognosis in diverse types of leukemia. However, the exact mechanism by which the splenic microenvironment alters responses to specific targeted therapies remains largely unexplored. We show that residual LOUCY cells isolated from the spleen microenvironment displayed reduced BCL-2 dependence, which was accompanied by decreased BCL-2 expression levels. Notably, this phenotype of reduced BCL-2 dependence could be recapitulated by using human splenic fibroblast coculture experiments and was confirmed in an in vitro chronic ABT-199 resistance model of LOUCY. Finally, single-cell RNA-sequencing was used to show that ABT-199 triggers transcriptional changes in T-cell differentiation genes in leukemic cells obtained from the spleen microenvironment. Of note, increased expression of CD1a and sCD3 was also observed in ABT199-resistant LOUCY clones, further reinforcing the idea that a more differentiated leukemic population might display decreased sensitivity toward BCL-2 inhibition. Overall, our data reveal the spleen as a site of residual disease for ABT-199 treatment in ETP-ALL and provide evidence for plasticity in T-cell differentiation as a mechanism of therapy resistance.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2–driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life–derived B1a cells, can be an effective therapeutic strategy to treat MCL.

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The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold’s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase (ASNS). Acute lymphoblastic leukemia (ALL) cells do not or minimally express ASNS which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have a significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anti-cancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have a very short half-life in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long lasting durable anti-leukemic effect between the standard-of-care PEG-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential.

Background: Some rare subgroups of leukemia cells harboring relapse-inducing genes were selected after chemotherapy.Tounravel intra-tumoral heterogeneity and selective drug-resistance, single-cell RNA sequencing (scRNA-seq) was already performed on many solid tumors and blood cancer to achieve the high-resolutiontranscriptome profiling on individual cells from a larger heterogeneous population. However,the comprehensive investigation on cancer heterogeneityduring cancer development at single-cell resolution is still rare. Aims: To identify diverse subsets and molecular characteristics of acute myeloid leukemia (AML) relapse Methods: Since single-cell suspension was obtainedfrom bone marrow of acute myeloid leukemia samples, we used the 10x GenomicsChromium platform to capture transcriptomes of singlecells on barcoded mRNA capture beadsfor massively parallel scRNA-seq. Data processing followed by the Cell Ranger software pipelineto demultiplex cellularbarcodes, and map reads to the genome and transcriptome hg38 using the STAR aligner.Uniquemolecular identifier (UMI) count matrix and quality control were performed using Seurat. The t-SNE map was calculated using Rtsne package Results: We analyzed transcriptome data from near 50K single leukemia bone marrow cells across 3 patients during newly diagnosed, complete remission and relapse stages. To define the landscape of cellular heterogeneity and its association with outcome in an unbiased manner, we performed unsupervised machine learning algorithm on near 50K single cells from leukemia bone marrow and identify one robust 14-cluster solution (from 0 to 13, Figure 1A) and the hallmark genes within each clusters (Figure 1B). The pattern exhibits distinct distribution on different stages (Figures 1C), indicating intra-tumoral heterogeneity during leukemia progression. Within cluster 0, the subgroups expressing such as LILRB2, TNFAIP2 or PTAFR were chemotherapy sensitive (Figure 2A). While the subgroups expressing such as APOC1, CDKN2A, KLF1 or GATA1 were chemotherapy resistant (Figure 2B). These chemotherapy resistant subgroups may play some key roles in leukemia relapse.

Glioblastoma remains a highly malignant and intrinsically resistant brain tumor. Despite intensive research through which numerous potential druggable targets were identified, virtually all clinical trials of the past 20 years failed to improve the outcome for the vast majority of GBM patients. However, the identification of small subgroups of patients that showed an exceptional response across several trials, implies that, when selected more carefully, some GBM patients could probably still benefit from these therapies. Identifying these patients requires that suitable biomarkers are identified. In this project, we reassessed the molecular mechanisms of ten actionable compounds (selected from previously failed trials but for which exceptional responders had been observed) in a set of carefully selected patient-derived cell lines that were sensitive/resistant to the selected therapies. Moreover, to deal with tumor heterogeneity, we used a multi-omic functional precision oncology approach, combining scRNA-seq and CyTOF, to identify drug-specific biomarkers by comparing control and treated samples at single-cell resolution. By subsequently correlating the molecular signatures to eventual cytotoxicity profiles, we could identify intrinsically responsive tumor cells at the single-cell level within hours following drug exposure. Overall, this work lays the foundation for an actionable functional diagnostic assay that could help to identify eligible GBM patients in future clinical trials.