Supplementary Figs. 1-17 with legends

Supplementary methods Supplementary Text: Wolinella succinogenes as a low L-glutaminase L-asparaginase Supplementary Figures: • Fig. S1. In vivo imaging showing that the L-glutaminase-diminished ErA-63Q and ErA-DM variants eliminate T-ALL LOUCY cells as effectively as the high L-glutaminase ErA-WT. • Fig. S2: In vivo imaging showing that the low L-glutaminase ErA-DM variant eliminates B-ALL SUP-B15 cells as effectively as the high L-glutaminase ErA-WT. • Fig. S3. In vivo imaging showing that the ultra-low L-glutaminase ErA-TM variant eliminates T-ALL LOUCY cells as effectively as the high L-glutaminase ErA-WT. • Fig. S4. In vitro sensitivity of two human ALL cell lines to L-asparaginases. • Fig. S5: Calibration between the in vivo imaging flux intensity and the measured %huCD45+ levels in peripheral blood (PB). • Fig. S6. In vivo imaging with signal-to-color range adjusted. • Fig. S7. Correlation between L-glutaminase activity and the reduction in mice activity. • Fig. S8. Correlation between L-glutaminase activity of the ErA variants and mice weight loss. • Fig. S9. ASNS qPCR data for the LOUCY cell line and 5 ALL patient samples. Data. Biostatistics on BLI and weight data.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFβ. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

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.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

BRCA2 (also known as FANCD1) is a core component

BRCA2 (also known as FANCD1) is a core component of the Fanconi pathway and suppresses transformation of immature T-cells in mice. However, the contribution of Fanconi-BRCA pathway deficiency to human T-cell acute lymphoblastic leukemia (T-ALL) remains undefined. We identified point mutations in 9 (23%) of 40 human T-ALL cases analyzed, with variant allele fractions consistent with heterozygous mutations early in tumor evolution. Two of these mutations were present in remission bone marrow specimens, suggesting germline alterations. BRCA2 was the most commonly mutated gene. The identified Fanconi-BRCA mutations encode hypomorphic or null alleles, as evidenced by their inability to fully rescue Fanconi-deficient cells from chromosome breakage, cytotoxicity and/or G2/M arrest upon treatment with DNA cross-linking agents. Disabling the tumor suppressor activity of the Fanconi-BRCA pathway is generally thought to require biallelic gene mutations. However, all mutations identified were monoallelic, and most cases appeared to retain expression of the wild-type allele. Using isogenic T-ALL cells, we found that BRCA2 haploinsufficiency induces selective hypersensitivity to ATR inhibition, in vitro and in vivo. These findings implicate Fanconi-BRCA pathway haploinsufficiency in the molecular pathogenesis of T-ALL, and provide a therapeutic rationale for inhibition of ATR or other druggable effectors of homologous recombination.

AbstractPurpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

Abstract T-cell acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors that result from the malignant transformation of T-cell progenitors. Due to intensified chemotherapy, the prognosis of T-ALL has gradually improved. Nevertheless, this clinical improvement is most pronounced in pediatric treatment protocols, whereas adult patients more often present with primary resistant or relapsed disease. Our aim is to develop new mouse models that mimic oncogenic lesions identified in human disease. These mouse models are critically required to further enhance our knowledge on the molecular mechanisms that drive T-cell leukemogenesis. Moreover, they serve as important preclinical models to evaluate new therapeutic strategies for the treatment of human T-ALL. Here, we generated a fast method for embryonic stem cell (ESC) targeting and mouse chimera production. We constructed gateway-compatible vectors that allow tailor-made targeting vector design, including conditional expression of a transgene combined with an eGFP/luciferase reporter from the ROSA26(R26)-promoter. The final targeting vector is recombination-mediated cassette exchange (RMCE)-compatible and can be inserted in a genetically engineered R26-locus via RMCE. Correct integration of the incoming construct reactivates the NeoR gene and results in up to 100% ESC targeting efficiencies. Using our technology, we generated R26-based conditional knock-in mouse models for putative oncogenes that have previously been implicated in T-ALL disease biology. More specifically, the MYB leucine zipper transcription factor is aberrantly activated in a subset of T-ALL patients through T-cell receptor driven translocations (t(6;7)(q23;q34)) or genomic duplications of the MYB locus itself. To study the in vivo roles of cMyb in the pathogenesis of T-ALL, we used the above-mentioned genomic engineering technology to generate cMyb conditional knock-in mouse models. These mice were crossed with VaviCre mice to obtain hematopoietic specific expression of Myb and the EGFP/luciferase from the ROSA26-promoter. We demonstrated that forced expression of Myb in hematopoietic precursors is sufficient to drive T-cell leukemia in mice. To allow comparative analysis of tumors with and without T-cell specific Myb expression, we also genetically deleted the tumor suppressor Pten in these mice and found that cMyb expression synergizes with Pten deletion and resulted in fully penetrant and accelerated T-ALL formation. These data demonstrate an important role for Myb in the pathologenesis of T-ALL. Finally, we use this novel murine T-ALL model to find new therapies for T-ALL. Importantly, the tumor cells from the cMyb knock-in mice are luciferase positive and are therefore suitable for in vivo drug testing using bioluminescence. Because cMYB activation drives an oncogenic transcription program by regulation of super-enhancer activity in human T-ALL will test drugs that impede Myb protein stability or Myb-mediated transactivation. Citation Format: Tim Pieters, Sara TSas, Beatrice Lintermans, Sofie Peirs, Filip Mathijssens, Jody Haigh, Geert Berx, Steven Goossens, Pieter Van Vlierberghe. Fast and efficient generation of conditional ROSA26-based mouse models that recapitulate oncogene activation in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A08.

Author(s): Milani, Gloria; Matthijssens, Filip; Van Loocke, Wouter; Durinck, Kaat; Roels, Juliette; Peirs, Sofie; Thenoz, Morgan; Pieters, Tim; Reunes, Lindy; Lintermans, Beatrice; Vandamme, Niels; Lammens, Tim; Van Roy, Nadine; Van Nieuwerburgh, Filip; Deforce, Dieter; Schwab, Claire; Raimondi, Susana; Dalla Pozza, Luciano; Carroll, Andrew J; De Moerloose, Barbara; Benoit, Yves; Goossens, Steven; Berx, Geert; Harrison, Christine J; Basso, Giuseppe; Cave, Helene; Sutton, Rosemary; Asnafi, Vahid; Meijerink, Jules; Mullighan, Charles; Loh, Mignon; Van Vlierberghe, Pieter

Abstract Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL. Significance: A new l-asparaginase–based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549–60. ©2018 AACR.

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1. These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive class of hematologic tumors caused by malignant transformation and abnormal proliferation of T-cell progenitors. T-ALL represents 10-15% of pediatric ALL and, despite the improved survival rate during the last decades, 20% of cases still experience relapsed and therapy failure. MYC serves as a pivotal oncogene in the pathogenesis of T-ALL that can be activated through a number of direct and indirect mechanisms. For example, a rare subgroup of primary T-ALL (~1%) is characterized by T cell receptor (TCR) driven translocations that cause massive activation of the MYC oncogene and present an unfavorable prognosis, rapid disease progression and poor response to conventional therapy. Here, we performed detailed molecular genetic characterization of an extensive series of primary T-ALL that present TCR driven MYC translocations and evaluated new therapeutic strategies for this poor prognostic subtype of human leukemia. Molecular genetic characterization of a cohort of 28 pediatric T-ALL patients carrying the t(8;14)(q24;q11) translocation was performed by copy number profiling, gene expression and mutational screening. Xenograft experiments with primary patient material were used to evaluate the therapeutic relevance of JQ1 in the context of MYC translocation positive T-ALL. Copy number analysis of MYC translocated T-ALL cases revealed loss of the previously reported T-ALL tumor suppressor genes PTEN (18%), CDKN2A/B (68%) and LEF1 (7%). Moreover, about 30% of patients harbored genomic deletions that cause aberrant activation of the SIL-TAL1 or LMO2 oncogenes. Interestingly, sequencing analysis revealed no aberrations in NOTCH1 or FBXW7 and a high number (30%) of loss-of-function mutations targeting PTEN. Therefore, TCRAD-MYC translocation positive T-ALL seems to represent a NOTCH1 independent subtype of leukemia that is frequently addicted to activated PI3K/AKT signaling. In line with this notion, the t(8;14)(q24;q11) positive T-ALL cell lines KE-37 and MOLT16 lack NOTCH1/FBXW7 mutations but both present loss of PTEN. Indeed, these tumor lines show aberrant pAKT activation but lack enhanced NOTCH1 activity at the protein level. In accordance with the frequent identification of TAL1 and/or LMO2 rearrangements, genome-wide transcription profiling analysis revealed that TCRAD-MYC positive T-ALL showed a uniform gene expression signature reminiscent of late cortical thymocyte T-ALL that express CD4, CD8 and membrane CD3. Importantly, gene set enrichment analysis revealed a negative enrichment of NOTCH1 target genes in MYC translocated late cortical T-ALLs, including NOTCH3, HES4, DLL4, PTCRA and DTX1. From a therapeutic perspective, in vitro drug sensitivity screening showed that MYC rearranged T-ALL cell lines were amongst the most sensitive to JQ1 treatment (IC50 values: MOLT16, 199nM; KE37, 497nM). MYC mRNA and protein downregulation were confirmed after JQ1 treatment for both the lines. Moreover, in vivo drug treatment experiments using xenografts generated from primary t(8;14)(q24;q11) positive T-ALL patients cells showed the in vivo activity of the JQ1 against this aggressive subtype of human T-ALL, as evaluated by percentage of leukemic blasts in blood or bone marrow and spleen size. Given the high frequency of genetic abnormalities targeting the PI3K/AKT pathway in these NOTCH1 independent human T-ALL, we are currently evaluating the synergistic activity of JQ1/MK-2206 combination therapy using the same in vivo model systems. In this study, we identified TCRAD-MYC rearranged T-cell leukemia as a novel subclass of NOTCH1-independent late cortical T-ALL that frequently depend on aberrant PI3K/AKT signaling. Moreover, we showed that JQ1 might serve as a promising new therapeutic strategy for the treatment of this high-risk subtype of pediatric leukemia. Citation Format: Gloria Milani, Kaat Durinck, Filip Matthijssens, Sofie Peirs, Tim Pieters, Lindy Reunes, Beatrice Lintermans, Niels Vandamme, Tim Lammens, Yunlei Li, Claire Schwab, Susana Raimondi, Barbara De Moerloose, Yves Benoit, Geert Berx, Christine Harrison, Giuseppe Basso, Helene Cavé, Rosemary Sutton, Vahid Asnafi, Charles Mullighan, Jules Meijerink, Mignon Loh, Pieter Van Vlierberghe. Genetic characterization and therapeutic targeting of MYC translocated pediatric T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B21.

Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.