The Aurora kinase/β-catenin axis contributes to dexamethasone resistance in leukemia

Glucocorticoids, such as dexamethasone and prednisolone, are widely used in cancer treatment. Different hematological malignancies respond differently to this treatment which, as could be expected, correlates with treatment outcome. In this study, we have used a glucocorticoid-induced gene signature to develop a deep learning model that can predict dexamethasone sensitivity. By combining gene expression data from cell lines and patients with acute lymphoblastic leukemia, we observed that the model is useful for the classification of patients. Predicted samples have been used to detect deregulated pathways that lead to dexamethasone resistance. Gene set enrichment analysis, peptide substrate-based kinase profiling assay, and western blot analysis identified Aurora kinase, S6K, p38, and β-catenin as key signaling proteins involved in dexamethasone resistance. Deep learning-enabled drug synergy prediction followed by in vitro drug synergy analysis identified kinase inhibitors against Aurora kinase, JAK, S6K, and mTOR that displayed synergy with dexamethasone. Combining pathway enrichment, kinase regulation, and kinase inhibition data, we propose that Aurora kinase or its several direct or indirect downstream kinase effectors such as mTOR, S6K, p38, and JAK may be involved in β-catenin stabilization through phosphorylation-dependent inactivation of GSK-3β. Collectively, our data suggest that activation of the Aurora kinase/β-catenin axis during dexamethasone treatment may contribute to cell survival signaling which is possibly maintained in patients who are resistant to dexamethasone.

Identification of Genetic Vulnerabilities and Synthetic-Lethal Targets in NSD2-High Multiple Myeloma

The histone methyltransferase NSD2 is overexpressed in 15-20% of multiple myeloma (MM) patients due to the t(4;14) chromosomal translocation. NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. Despite strong efforts by academia and industry, there remains no tool compound or drug that inhibits NSD2 enzymatic activity. Therefore we explored the molecular and biochemical consequences of NSD2 overexpression in MM cells and investigated genetic vulnerabilities associated with high NSD2 levels as well as synthetic-lethal drug-gene interactions in t(4;14)-positive MM cells. We utilized the well-studied isogenic pair of human MM cells derived from the t(4;14)-positive KMS-11 cell line, where NSD2 is disrupted by knocking out either the translocated overexpressed allele (TKO; NSD2-low) or the wild-type non-translocated allele (NTKO; NSD2-high). Untargeted metabolomic profiling of TKO and NTKO cells revealed that NSD2 overexpression substantially alters nucleotide metabolism. NSD2-high cells exhibited an increase in purine synthesis and a decrease in pyrimidine synthesis. In addition, a genome-wide loss-of-function CRISPR gene editing screen using the 76,000 guide Brunello library uncovered genes differentially essential between NSD2-high and low MM cells. Intriguingly, one of the genes whose disruption is selectively lethal in NSD2-high cells encodes Adenylate Kinase 2 (AK2), an enzyme involved in purine metabolism, indicating that defective nucleotide metabolism associated with increased NSD2 expression introduces genetic vulnerabilities that can be therapeutically exploited. We further explored liabilities that can improve therapeutic outcomes in t(4;14) MM. A genome-wide CRISPR screen was performed in KMS-11 MM cells to identify mechanisms of sensitivity and resistance to dexamethasone, a steroid commonly used in MM treatment. As expected, inactivating the gene encoding the glucocorticoid receptor (GR) resulted in remarkable dexamethasone tolerance. We then identified and validated multiple genes/pathways that can alter response of MM cells to dexamethasone when disrupted. For example, inactivation of interleukin 10 (IL10) signaling by disrupting either subunit of the IL10 receptor or components of the downstream JAK/STAT pathway considerably enhances dexamethasone sensitivity. In addition, disruption of many components of heparan sulfate or glycosaminoglycans synthesis pathways, whose targeting has been previously shown to increase sensitivity to conventional MM chemotherapeutic agents, increases susceptibility to dexamethasone. By contrast, genetic perturbations leading to dexamethasone resistance correspond to GR co-chaperones including FKBP4 and PTGES3 and transcriptional coactivators like the nuclear receptor transactivator 1 (NCOA1). Interestingly, disruption of genes encoding proteins implicated in RNA stability and translation such as the N6-methyladenosine (m6A)-containing RNA binding protein YTHDF2 and the PAN2-PAN3 deadenylase complex results in dexamethasone resistance. How these proteins affect the expression of pro or anti-apoptotic genes in response to dexamethasone is under investigation. Our work reveals insight into novel molecular-based treatment options for t(4;14) MM that are independent on direct NSD2 inhibition which remains unsuccessful. Disclosures No relevant conflicts of interest to declare.

Whole-Genome CRISPR Screen Reveals the Mechanism of Relapse in Patient-Derived Cells Representing High-Risk Paediatric ALL

Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome. In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled 'Brunello' library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs. Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone. Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples. In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation. Disclosures Vormoor: Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.