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Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of precursor B or T cells (B- or T-ALL). Novel immunotherapies are urgently needed to reduce polychemotherapy-related toxicities and to target relapsed/refractory disease. The Interleukin 7 receptor (IL7R) plays a pivotal role in the development of ALL through mutations leading to constitutive activation of the oncogenic IL7R pathway or through overexpression of the receptor. The IL7Rα chain (CD127)-targeting antibody OSE-127 is a full antagonist of the IL7R pathway with an excellent safety profile (NCT03980080), currently evaluated in phase 2 trials in inflammatory and autoimmune diseases (NCT04882007, NCT04605978). We previously reported in vivo efficacy of OSE-127 in CD127+ ALL patient-derived xenografts (PDXs) and synergy with standard of care polychemotherapy, but we had not evaluated CD127 protein expression in ALL patient cells. Moreover, the mechanism of action underlying OSE-127’s anti-leukemic activity remained to be fully elucidated. Here we show that CD127 is expressed by >84% ALL patients and that OSE-127 efficiently targets CD127 not only via its IL7R antagonist activity but also through macrophage-mediated antibody dependent phagocytosis (ADCP). First, CD127 surface expression was prospectively measured by flow cytometry in 372 diagnostic ALL patient samples. We detected CD127-positivity (defined as ≥10% CD127+ blasts) in 84.4% (314/372) cases, of which 39.5% (147/372) were CD127hi (≥ 50% CD127+ blasts). CD127 expression was higher in T- than in B-ALL. Among B-ALL, highest expression was detected in E2A-PBX1+ and BCR-ABL-like ALLs. Mechanistically, OSE-127 blocked STAT5 phosphorylation in IL7-responsive ALL-PDX cells, leading to decreased cell survival and proliferation. Surprisingly, we detected high in vivo efficacy of OSE-127 therapy in IL7-unresponsive PDXs (e.g., unable to induce phosphorylation of STAT5 upon ex vivo IL7 stimulation or with IL7R mutation-induced constitutive phosphorylation of STAT5). We uncovered that OSE-127 induced macrophage-mediated ADCP of CD127+ ALL cells in vitro. Accordingly, treatment of IL7-unresponsive, but OSE-127-sensitive models with an OSE-127-LALAPG variant (conserving IL7R-antagonistic properties but lacking ADCP activity due to Fc-inactivating mutations) did not impact leukemia development, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127. In fact, ADCP levels induced by OSE-127 treatment in vitro correlated with its capacity to reduce ALL blasts in the blood of PDX mice in vivo. Altogether, through its dual mode of action, OSE-127 may represent a powerful novel immunotherapy option for ALL patients, including cases with dysregulated IL7R signaling, particularly in combination with standard of care polychemotherapy. Citation Format: Irene Baccelli, Lennart Lenk, Anna Laqua, Dorothee Winterberg, Anna Dietterle, Frederique Corallo, Julien Taurelle, Emma Narbeburu, Beat Bornhauser, Jean-Pierre Bourquin, Fotini Vogiatzi, Simon Raffel, Martin Schrappe, Gunnar Cario, Monika Brüggemann, Denis M Schewe, Nicolas Poirier. CD127 is expressed by acute lymphoblastic leukemias and is efficiently targeted by the IL7R-antagonist OSE-127 through macrophage-mediated antibody dependent phagocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2957.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL. Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg. Cancer Res; 78(22); 6497–508. ©2018 AACR.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional co-activator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due in part to upregulation of alternative pathways including WNT through a mechanism suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small molecules inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.

The identification of molecular determinants that regulate sensitivity to specific agents is essential for the development of new therapeutic approaches in cancer. We have earlier shown that a subset of refractory acute lymphoblastic leukaemia (ALL) samples respond to SMAC-mimetic (SM) induced IAP depletion by concurrently inducing RIP1-dependent apoptosis and necroptosis. Comparative gene expression profiling indicated a correlation of sensitivity to SM with the expression of TNF receptor 2 (TNFR2) in primary ALL. Using an independent cohort of primary chemotherapy-resistant ALL samples, we found that presence of high TNFR2 expression identified by qPCR predicted ex vivo-sensitivity to SM. High TNFR2 levels also correlated with higher expression of TNFR1. Deletion of either TNFR1 or TNFR2 using CRISPR/Cas9 in primary ALL conferred resistance to treatment with SM, indicating that TNFR1 and 2 are both functionally required for cell death. Concomitant with an important role for TNFR2 in the response to SM, the overexpression of TNFR2 leads to increased sensitivity to TNF through increased activation of the TNFR1/RIP1 death axis. On the mechanistic level, SM induced recruitment of RIP1 to TNFR1, which was abolished in cells deficient for TNFR2. Taken together, our data reveal a novel function of TNFR2 in cell death signalling, as TNFR2 predicts sensitivity to SMAC mimetics and plays a key role in modulating a switch from RIP1-controlled cell survival to cell death. Citation Format: Julia Aguadé-Gorgorio, Scott McComb, Cornelia Eckert, Maria Pamela Dobay, Gunnar Cario, Caterina Mezzatesta, Arend Von Stackelberg, Martin Stanulla, Schrappe Martin, Jean-Pierre Bourquin, Beat C. Bornhauser. TNF receptor 2 is essential for RIP1-dependent cell death in refractory leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2017-4321

Tumor cells evolve adaptive mechanisms to survive hypoxia, nutrient deprivation, oxidative or genotoxic stress to ultimately drive tumor progression (Sorensen et al. 2015). We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Lymphocytes, in particular B Lymphocytes are adapted to hypoxic environments from their very beginning. They are destined to travel from the hypoxic bone marrow via normoxic peripheral blood to hypoxic lymph nodes. They thus are specialized in glycolysis to compensate the lack of oxidative phosphorylation under hypoxic conditions. Leukemic counterparts of B-lymphocytes exploit these features for their survival. Glucose-derived metabolites control the nuclear activity of the transcription factor MondoA. Here we report on the expression of MondoA in common B-cell acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Methods. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA. Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. Results. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rhabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in mice after MondoA KD. Further microarray analysis revealed an enrichment of glycolytic and hypoxia response gene sets by MondoA. Moreover, HIF1a induction under hypoxia required MondoA. We demonstrate that hypoxia facilitates vincristine resistance of cALL. MondoA is induced under hypoxia and confers cALL cells chemotherapeutic resistance. Tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response, in particular by induction of HIF1a. Citation Format: Alexandra A. Sipol, Thomas G. Grunewald, Juliane Schmaeh, Monique L. den Boer, Rebeca Alba Rubío, Michaela Baldauf, Caroline Wernicke, Martin Horstmann, Gunnar Cario, Günther Richter, Stefan Burdach. Metabolic stress sensor MondoA mediates in vivo aggressiveness of common ALL by induction of HIF1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2017-4515

Dysregulation of apoptosis constitutes a general mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death during chemotherapy. We have assessed the potential of SMAC mimetic compounds (SM) to eradicate drug resistant ALL by blocking the pro-survival cellular inhibitor of apoptosis proteins (cIAPs). On the basis of a large xenograft bank of primary ALL samples, obtained by xenotransplantation in immunodeficient NSG mice, we detected exquisite sensitivity to SM in vitro in a third of the cases tested, including drug-resistant and relapsed samples. Potent in vitro activity was highly predictive of in vivo efficacy in the xenograft model. Treatment of transplanted mice with SM not only delayed leukemia progression, but also completely eliminated leukemia burden in established disease for several weeks following treatment. SM activity required the presence of RIP1, since lentiCRISPR mediated gene knockout of RIP1 in patient-derived ALL cells blocked SM-induced cell death in vivo and in vitro. In contrast, RIP1 loss had no significant impact on the response to standard antileukemic therapies, supporting a view that RIP1-dependent death pathways are not likely to be selected against by front line chemotherapy. Furthermore, using a multicolor lentiCRISPR approach for simultaneous disruption of multiple genes, we found that downstream of RIP1, the response to SM was dependent on simultaneous execution of apoptosis and necroptosis, since genetic targeting of both cell death pathways was required to block SM-induced death. Upstream of RIP1, expression levels of TNF receptor II (TNFR2) in primary ALL correlated with sensitivity to SM, and genetic disruption of TNFR2 conferred resistance to SM treatment in vivo. Thus, SM treatment circumvents drug resistance through parallel activation of RIP1-dependent apoptosis and necroptosis, providing a strong rationale for application in salvage treatment of refractory childhood ALL. Citation Format: Júlia Aguadé-Gorgorió, Scott McComb, Gunnar Cario, Martin Stanulla, Cornelia Eckert, Jean-Pierre Bourquin, Beat C. Bornhauser. In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3548.

Oncogene addiction provides ideal targets for immunotherapy. We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Here we report on the expression of MondoA in common acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Given the non-accessibility of transcription factors by drugs or chimeric antigen receptor transgenic T cells (CARs), we tested the targetability of MondoA by allo-restricted, peptide specific T cells. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA (shRNA). Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MondoA specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MondoA peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MondoA peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon gamma (IFg) and granzyme B assays with six MondoA+ leukemia lines (A2+, A2-). Off target effects of MondoA specific T-cell clones were assessed by IFg reactivity against the MondoA expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in treated mice after MondoA KD. Further microarray analysis revealed an induction of aerobic glycolysis switch genes and hypoxia-response by MondoA. Consequently, HIF1A stabilization required MondoA expression and tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. Therapeutically, MondoA-derived peptide antigens and A2+ cALL lines were successfully recognized and killed by specific, allo-restricted CD8+ T cells. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response. Moreover, we identified MondoA as a promising target for immunotherapy of cALL. Citation Format: Alexandra Sipol, Thomas G. P. Grunewald, Juliane Schmaeh, David Schirmer, Monique L. den Boer, Rebeca Alba Rubío, Michaela Baldauf, Caroline Wernicke, Hans-Jochem Kolb, Martin Horstmann, Gunnar Cario, Guünther Richter, Stefan Burdach. MondoA mediates in vivo aggressiveness of common ALL and may serve as a T-cell immunotherapy target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2462.

Novel treatment approaches are needed for patients with acute lymphoblastic leukemia (ALL) who respond poorly to current therapies. The genotypic diversity identified recently by next generation sequencing technologies within ALL calls for individualized novel strategies. However, functional correlations of oncogenic lesions with drug response profiles are ill defined for ALL. We have established an imaging-based cell viability analysis platform to generate drug response profiles for primary patient-derived ALL samples co-cultured with mesenchymal stroma cells, expecting to derive functional information directly from individual patient samples. Such response profiles may mirror perturbations in relevant cellular programs that could be exploited therapeutically. Our pipeline integrates high-content screening, newly developed bioinformatics tools and biochemical approaches. We screened a library of 65 compounds for activity in 37 precursor B-ALL and 23 T-ALL samples including refractory cases. Cross-sample comparisons revealed that cells from relapsed refractory patients showed a more resistant phenotype for most of the drugs. While only a few agents including genotoxic drugs showed activity across all samples, we detected selective activity of given drugs that distinguish patient sample groups. MLL-rearranged and TCF3-HLF-positive ALL samples as well as a subgroup of T-ALL cases were highly sensitive to the BCL-2 specific BH3-mimetic ABT-199 suggesting BCL-2 dependency for these cases. Multiparametric analyses of in vitro responses predicted in vivo activity of ABT-199 in xenografted mice. Moreover, we could identify synergistic activity of ABT-199 with clinical and preclinical compounds, such as topotecan or epigenetic modifiers. Our drug response profiling pipeline contributes to the identification of distinct vulnerabilities in leukemia and may facilitate the selection of candidate drugs for further development into clinical application. Citation Format: Viktoras Frismantas, Anna Rinaldi, Maria Pamela Dobay, Salome Higi, Sabrina Eugster, Blerim Marovca, Peter Horvath, Mauzro Delorenzi, Joachim Kunz, Obul R. Bandapalli, Gunnar Cario, Martin Stanulla, Andreas E. Kulozik, Martina Muckenthaler, Cornelia Eckert, Thomas Radimerski, Jean-Pierre Bourquin, Beat C. Bornhauser. Drug response profiling to inform individualized treatment approaches in high risk leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2015-493

Xenotransplantation of primary leukemia cells constitutes an attractive strategy to model disease for translational research, but the consequences of selective pressure on leukemia cells need to be clarified. Here we evaluate the phenotypic and genetic stability of primograft samples that were established by xenotransplantation of ALL cells from a group of patients with very high-risk precursor B-cell ALL, characterized by the persistence of minimal residual disease (VHR-ALL by MRD) after an intensive induction and consolidation therapy. Phenotypic properties remained unchanged, with concordant immunophenotypes in 9-10/10 leukemia-associated markers after up to 5 passages in Nod/scidIL2Rgammanull (NSG) mice and with stable drug response profiles in vitro on bone marrow stroma co-cultures. In 4/6 cases, 100 unsorted ALL cells were sufficient to reconstitute the leukemia. From up to 5 recurrent copy number abnormalities (CNA) detected in diagnostic samples, most were maintained in primografts. Changes in CNAs upon transplantation in NSG mice were few with 0-2 new lesions per case, mostly occurring during the first xenotransplantation. At the single cell level, the pattern of deletions in the CDKN2A/B locus revealed distinct subsets of ALL cells, which could reproducibly be tracked in primografts. These results provide unequivocal data for the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for the use of this model for translational research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2011-5330

Relapse of acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related childhood mortality. Persistence of significant levels of minimal residual disease after almost 6 months of treatment identifies a group of patients with very high risk of relapse (VHR-ALL). Prognostic markers are not available to identify this subgroup at diagnosis. These VHR-ALL patients would qualify for new experimental approaches as part of their initial therapy. Here we present an extensive analysis of the cell surface glycoproteome of ALL cells from 8 VHR-ALL patients and from a corresponding set of ALL patients with good clinical outcome. Given the very limited amount of primary diagnostic material available for research, we have amplified primary human ALL cells in our established xenotransplantation model, using immunodeficient NSG mice. This amplification of primary human material allowed us for the first time to generate normally rare human leukemic cells in numbers sufficient for proteomic studies. We have optimized and extended the mass spectrometry-based Cell Surface Capturing (CSC) technology with complementary enrichment strategies, to increase the protein sequence coverage. Roughly 2500 candidate cell surface proteins were identified. 40% of the candidates were predicted by the TMHMM algorithm to contain a transmembrane domain or a GPI-anchor, also including 174 known CD markers. The immunophenotype analysis of leukemia associated surface markers by flow cytometry at diagnosis was recapitulated in our proteomic dataset in all cases. A semi-quantitative spectral counting approach identified a subset of 200 proteins consistently expressed on most ALL samples and a subset of 60 proteins preferentially detected on the surface of VHR-ALL samples. A subgroup of VHR-ALL, which represented 50% of the analysed cases, was characterized by high expression of members of the vanin (VNN) protein family. Flow cytometry data of VNN-2 from xenografted samples correlated with the semiquantitative estimation of protein levels by the CSC technology. In an independent cohort of 20 ALL samples, we did not detect VNN-2 expression on ALL cells from standard risk patients, while we detected VNN-2 expression in 4 out of 8 cases of high risk or relapsed patients. Anti-VNN-2 antibodies stained the ALL cells homogeneously, indicating that VNN-2 expression could mark the entire population of leukemia cells. Since vanin family proteins have been proposed to mediate interactions between hematopoietic cells and their microenvironment, our findings also provide the basis to evaluate the functional role of vanins in ALL. Our data provide an unprecedented view at the cell surface landscape of the most refractory leukemia cases and identify subsets of cell surface proteins that could be used for diagnosis or therapeutic intervention in this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4358.