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A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

A common problem in the study of human malignancy is the elucidation of cancer driver mechanisms associated with recurrent deletion of regions containing multiple genes. Taking B-cell acute lymphoblastic leukaemia (B-ALL) and large deletions of 6q [del(6q)] as a model, we integrated analysis of functional cDNA clone tracking assays with patient genomic and transcriptomic data, to identify the transcription factors FOXO3 and PRDM1 as candidate tumour suppressor genes (TSG). Analysis of cell cycle and transcriptomic changes following overexpression of FOXO3 or PRDM1 indicated that they co-operate to promote cell cycle exit at the pre-B cell stage. FOXO1 abnormalities are absent in B-ALL, but like FOXO3, FOXO1 expression suppressed growth of TCF3::PBX1 and ETV6::RUNX1 B-ALL in-vitro. While both FOXOs induced PRDM1 and other genes contributing to late pre-B cell development, FOXO1 alone induced the key transcription factor, IRF4, and chemokine, CXCR4. CRISPR-Cas9 screening identified FOXO3 as a TSG, while FOXO1 emerged as essential for B-ALL growth. We relate this FOXO3-specific leukaemia-protective role to suppression of glycolysis based on integrated analysis of CRISPR-data and gene sets induced or suppressed by FOXO1 and FOXO3. Pan-FOXO agonist Selinexor induced the glycolysis inhibitor TXNIP and suppressed B-ALL growth at low dose (ID50

The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.Statement of SignificanceWe demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.

The methyltransferase, KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis we have performed a microarray study in a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and Gene Ontology analysis revealed apoptosis and DNA damage signaling are up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase dependent manner to modulate both histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilized as a biomarker for effective therapeutic targeting.

The androgen receptor (AR) is a key driver of prostate cancer development. Antiandrogens effectively inactivate the AR, but subsequent AR reactivation progresses the disease to castrate-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-V) that function unchallenged by current AR-targeted therapies are key drivers of CRPC. Currently, very little is known about the regulation of AR-Vs at the chromatin level. Here, we show that the pioneer factor GATA2 is a critical regulator of AR-Vs. Furthermore, we demonstrate that the GATA2 cistrome in CRPC shares considerable overlap with bromodomain and extraterminal (BET) proteins and is codependent for DNA binding. GATA2 activity is compromised by BET inhibitors, which attenuates the pioneering role of GATA2 in CRPC. In all, this study indicates that GATA2 is a critical regulator of AR-V–mediated transactivation and is sensitive to BET inhibitors, signifying these agents may be efficacious in patients with CRPC which overexpress GATA2.Implications:We have defined novel mechanisms of AR-V and GATA2 regulation in advanced prostate cancer that could be therapeutically exploited.

siRNA sequences

Supplementary figure S2: A) Western blot shows optimisation of anti-WIP1 antibody (F-10) using siRNA mediated knockdown in MCF-7 cells. Following 24 and 48 hours of siRNA mediated knockdown conditions, MCF-7 cells were treated with 10Gy IR, lysates were collected after 4 hours and analysed by western blotting. All the bands shown were knocked down in response to four different WIP1 targeting siRNA constructs (WIP1.1-4) compared to the universal control siRNA (Cont. siRNA). WIP1 knockdown also resulted in higher and persistent pp53Ser15 most notably in response to the WIP1.2 siRNA construct. WIP1.1 Sense: GUGCCAUAGUAAUCUGCAU; WIP1.2 Sense: GGUGUAGUCAUACCCUCAA; WIP1.3 Sense: GCCCUUCCUAUAAUAGUCA; WIP1.4 Sense: UUGGCCUUGUGCCUACUAA Universal control Sense: GCGCGCUUUGUAGGAUUCG. B) Nutlin-3 and RG7388 SRB growth inhibition curves in MCF-7 cells in the presence and absence of 2.5µM GSK2830371. C) On the left side two independent repeats of the western blot in Figure 2A showing degradation of WIP1 protein in MCF-7 cells over time following treatment with 2.5µM GSK2830371. Full-length WIP1 band optical density (OD) was normalised to actin OD over time.

qPCR primers

ChIP primers

Supplementary Table S1: A & B) List of genes with significantly increased mRNA levels in NGP cells 4 hours following treatment with 75nm RG7388 (GI50 in NGP cells) + 2.5µM GSK2830371. Genes were ordered based on p-value following Benjamini-Hotchberg correction for multiple testing. OR: Odds ratio.

S1: GATA2 depletion down-regulates AR-V target genes S2: Chromatin occupancy of AR-Vs is not perturbed by GATA2 depletion S3: Overlapping GATA2 and AR-V transcriptomes S4: Gene ontology of GATA2 and AR-V transcriptomes S5: Assessing GATA2 and BET protein interplay in prostate cancer cell lines S6: Global cistromic analysis of GATA2 S7: Overlapping cistromes of GATA2 and BET family members S8: Assessing overlap of GATA2 and BET protein-regulated transcriptomes S9: GATA2 regulates splicing activity in prostate cancer cells

Hepatocellular carcinoma (HCC), the most common type of liver cancer, has very poor outcomes. Current therapies often have low efficacy and significant toxicities. Thus, there is a critical need for the development of novel therapeutic approaches for HCC. We have developed a novel bioinformatics pipeline, which integrates genome-wide DNA methylation and gene expression data, to identify genes required for the survival of specific molecular cancer subgroups but not normal cells. Targeting these genes may induce cancer-specific “synthetic lethality”. Initially, five potential HCC molecular subgroups were identified based on global DNA methylation patterns. Subgroup-2 exhibited the most unique methylation profile and two candidate subtype-specific vulnerability or SL-like genes were identified for this subgroup, including TIAM1, a guanine nucleotide exchange factor encoding gene known to activate Rac1 signalling. siRNA targeting TIAM1 inhibited cell proliferation in TIAM1-positive (subgroup-2) HCC cell lines but had no effect on the normal hepatocyte HHL5 cell line. Furthermore, TIAM1-positive/subgroup-2 cell lines were significantly more sensitive to the TIAM1/RAC1 inhibitor NSC23766 compared with TIAM1-negative HCC lines or the normal HHL5 cell line. The results are consistent with a synthetic lethal role for TIAM1 in a methylation-defined HCC subgroup and suggest it may be a viable therapeutic target in this subset of HCC patients.

Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC.

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC-even in the absence of progressive injury and fibrosis. ispartof: SCIENTIFIC REPORTS vol:11 issue:1 ispartof: location:England status: published

T-cell acute lymphoblastic leukemia (T-ALL) is frequently characterized by glucocorticoid (GC) resistance, which is associated with inferior outcomes, thus highlighting the need for novel therapeutic approaches for GC-resistant T-ALL. The pre-T-cell receptor (pTCR)/TCR signaling pathways play a critical role in cell fate decisions during physiological thymocyte development, with an interplay between TCR and glucocorticoid receptor (GR) signaling determining the T-lymphocyte selection process. We performed an shRNA screen in vitro and in vivo in T-ALL cell lines and patient-derived xenograft (PDX) samples to identify vulnerabilities in the pTCR/TCR pathway and identified a critical role for the lymphocyte cell-specific kinase (LCK) in cell proliferation. LCK knockdown or inhibition with dasatinib (DAS) caused cell cycle arrest. Combination of DAS with dexamethasone (DEX) resulted in significant drug synergy leading to cell death. The efficacy of this drug combination was underscored in a randomized phase II-like murine trial, recapitulating an early phase human clinical trial. T-ALL expansion in immunocompromised mice was significantly impaired using this drug combination, compared to mice receiving control vehicle or single drug treatment, highlighting the immediate clinical relevance of this drug combination for high-risk T-ALL patients. Our results thus provide a strategy to improve the efficacy of current chemotherapy platforms and circumvent GC resistance.

The fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of acute myeloid leukemia. Here we show that the RUNX1/RUNX1T1 oncogene is a regulator of alternative RNA splicing in leukemic cells. The comprehensive analysis of RUNX1/RUNX1T1-associated splicing events identifies two principal mechanisms that underlie the differential production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start site selection, and (ii) direct or indirect control of the expression of genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5’-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals alternative splicing as an important component of transcriptome re-organization in leukemia by an aberrant transcriptional regulator., The fusion gene RUNX1/RUNX1T1 is oncogenic in acute myeloid leukemia. Here, the authors show that the fusion gene alters the transcriptional landscape of the cells by changing the structure of the 5’UTR, altering isoform expression, and controlling the expression of splicing factors.

Background Chronic lymphocytic leukaemia (CLL) patients display a highly variable clinical course, with progressive acquisition of drug resistance. We sought to identify aberrant epigenetic traits that are enriched following exposure to treatment that could impact patient response to therapy. Methods Epigenome-wide analysis of DNA methylation was performed for 20 patients at two timepoints during treatment. The prognostic significance of differentially methylated regions (DMRs) was assessed in independent cohorts of 139 and 163 patients. Their functional role in drug sensitivity was assessed in vitro. Results We identified 490 DMRs following exposure to therapy, of which 31 were CLL-specific and independent of changes occurring in normal B-cell development. Seventeen DMR-associated genes were identified as differentially expressed following treatment in an independent cohort. Methylation of the HOXA4, MAFB and SLCO3A1 DMRs was associated with post-treatment patient survival, with HOXA4 displaying the strongest association. Re-expression of HOXA4 in cell lines and primary CLL cells significantly increased apoptosis in response to treatment with fludarabine, ibrutinib and idelalisib. Conclusion Our study demonstrates enrichment for multiple CLL-specific epigenetic traits in response to chemotherapy that predict patient outcomes, and particularly implicate epigenetic silencing of HOXA4 in reducing the sensitivity of CLL cells to therapy.

Introduction: Molecular diagnostic testing for lymphoproliferative disorders (LPDs) includes detection of clonal immunoglobulin (IG) and/or T cell receptor (TCR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. To date, laboratories still rely on subjective and labour-intensive technologies, e.g. karyotyping/FISH to detect complex genomic alterations. Whole Genome Sequencing (WGS) can detect all genomic alterations, but factors such as cost, computation/storage, DNA requirements and poor detection of clinically relevant subclonal mutations limits the routine implementation of WGS in clinical diagnostics. We have developed "KaryoSequencing" (KS), a novel approach that combines targeted deep-sequencing, using a targeted hybridisation capture NGS panel, for rare mutation and translocation detection with shallow WGS (sWGS) for genome wide copy number analysis, in a single test. Methods: KS was validated using 138 clinical samples from patients with acute lymphoblastic leukaemia (ALL) (n=46), chronic lymphocytic leukaemia (CLL) (n=46) and plasma cell myeloma (PCM) (n=46) samples from 3 UK laboratories. Samples underwent library preparation and hybridisation using the EuroClonality-NDC assay. A KS library for each sample was prepared by combining the pre-capture and post-capture libraries at an optimised ratio to enable high coverage (>500 x) at regions covered by the targeted panel while providing 0.5-1x coverage genome-wide. Forty-six KS libraries were pooled and sequenced per NovaSeq 6000 run, using a 200-cycle SP flow cell and a 100bp paired-end strategy. For analysis of targeted regions, somatic mutation calling was performed using VarDict and structural variants (rearrangements and translocations) were detected using ARResT/Interrogate. For analysis of large chromosomal copy number variation using sWGS, a modified version of QDNASeq/ACE with a window size of 50kb was performed. For sWGS analysis, bioinformatic exclusion of all target capture regions and panel-specific off-target regions was performed using a panel of 48 DNA samples from healthy individuals ran on the same KS protocol. Results: Analysis of genome-wide copy number by sWGS was concordant in 477 of 503 (95%) evaluable FISH tests, including precise detection of hyper and hypo-diploidy and other complex karyotypes. The performance of the targeted deep-sequencing component of the KS approach was assessed to ensure comparable performance to previously validated results for the EuroClonality-NDC. The EuroClonality-NDC detected a clonal IG rearrangement in 46/46 (100%) CLL cases and in 42/46 (91.3%) PCM cases. Clonality was detected in 43/46 (93.5%) and 44/46 (95.7%) cases at an IG and TCR locus respectively in ALL, a disease entity known to exhibit cross-lineage rearrangements. Overall, 44 translocations were detected in the 138 samples by the EuroClonality-NDC. FISH results were available for 32 of the 44 translocations detected by NGS and were concordant in 29/32 (91%). In two PCM samples NGS and FISH reported a different translocation; in the ALL sample, the FISH pattern showed an additional copy but not a split signal. Across the entire cohort of 138 samples, KaryoSequencing detected 190 mutations with a variant allele frequency ranging from 4.0 - 97.6%. Of the detected mutations, 69/190 (36.3%) had a VAF Conclusions: KaryoSequencing demonstrated >95% sensitivity and specificity for detection of gross genome-wide copy number alterations while retaining the analytical performance for detection of rearrangements, translocations, and mutations for the lymphoid-specific targeted regions of the EuroClonality-NDC assay. KS is a cost-effective and high-throughput integrated alternative to current diagnostic strategies in haematological malignancies and can be implemented in routine clinical practice. Disclosures Jenner: Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy; Takeda: Consultancy; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau. Gonzalez: Univ8 Genomics Ltd: Current holder of individual stocks in a privately-held company.

SUMMARYThe fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of the t(8;21)-positive acute myeloid leukemia. Here we show that this oncogene is a regulator of the alternative RNA splicing for a sub-set of genes in the leukemia cells. We found two primary mechanisms underlying changes in the production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start sites selection in target genes, and (ii) direct or indirect control of the expression of the genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5’-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals a novel layer of RUNX1/RUNX1T1-dependent transcriptome organization in t(8;21)-positive acute myeloid leukemia.

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53Ser15 phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non–growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor–induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53Ser15, known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53Ser15 and its potential use as a biomarker for response to this combination regimen. Mol Cancer Ther; 15(3); 379–91. ©2016 AACR.

SummaryThe translocation MLL-AF4 defines a high-risk subtype of acute leukaemia which, uniquely amongst MLL translocations, is almost exclusively associated with a pro-B lymphoid phenotype. However, the ability to switch lineage at relapse allowed interrogation of the cellular origin and lineage determinants of this leukaemia. The origin of MLL-AF4 lymphoid leukaemia was commonly within a multipotent precursor population. In contrast, relapse as MLL-AF4 AML occurs from either a multipotent progenitor or a committed pro-B leukaemic clone. Lineage switching results from the dysregulation of chromatin modifiers, including the nucleosome remodelling and deacetylation complex, NuRD, completely extinguishing the B lymphoid gene regulatory network, instead establishing a myeloid epigenotype. We propose that, in contrast to mixed phenotype acute leukaemias, lineage switch in MLL-AF4 leukemia is driven and maintained by a reorganised epigenome.

Summary Oncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML., Graphical Abstract, Highlights • An RNAi screen identifies CCND2 as a crucial transcriptional target of RUNX1/ETO • RUNX1/ETO promotes CCND2 expression by binding to an upstream element • CCND2 knockdown inhibits RUNX1/ETO-driven leukemic expansion in vitro and in vivo • RUNX1/ETO-expressing leukemic cells are highly sensitive to a CDK4/6 inhibitor, Using in vitro and in vivo screens to identify essential RUNX1/ETO transcriptional targets in AML, Martinez-Soria identify CCND2 as required for leukemia maintenance and self-renewal. Targeting this dependency using the CDK4/6 inhibitor palbociclib prolongs the survival of AML PDX models.

The TP53-MDM2-AR-AKT signalling network plays a critical role in the development and progression of prostate cancer. However, the molecular mechanisms regulating this signalling network are not completely defined. By conducting transcriptome analysis, denaturing immunoprecipitations and immunopathology, we demonstrate that the TP53-MDM2-AR-AKT cross-talk is regulated by the deubiquitinating enzyme USP12 in prostate cancer. Our findings explain why USP12 is one of the 12 most commonly overexpressed cancer-associated genes located near an amplified super-enhancer. We find that USP12 deubiquitinates MDM2 and AR, which in turn controls the levels of the TP53 tumour suppressor and AR oncogene in prostate cancer. Consequently, USP12 levels are predictive not only of cancer development but also of patient’s therapy resistance, relapse and survival. Therefore, our findings suggest that USP12 could serve as a promising therapeutic target in currently incurable castrate-resistant prostate cancer.

Metastatic castration resistant prostate cancer is one of the main causes of male cancer associated deaths worldwide. Development of resistance is inevitable in patients treated with anti-androgen therapies. This highlights a need for novel therapeutic strategies that would be aimed upstream of the androgen receptor (AR). Here we report that the novel small molecule anti-androgen, galeterone targets USP12 and USP46, two highly homologous deubiquitinating enzymes that control the AR-AKT-MDM2-P53 signalling pathway. Consequently, galeterone is effective in multiple models of prostate cancer including both castrate resistant and AR-negative prostate cancer. However, we have observed that USP12 and USP46 selectively regulate full length AR protein but not the AR variants. This is the first report of deubiquitinating enzyme targeting as a strategy in prostate cancer treatment which we show to be effective in multiple, currently incurable models of this disease.

The Androgen Receptor (AR) is a key molecule in the development, maintenance and progression of prostate cancer (PC). However, the relationship between the AR and co-regulatory proteins that facilitate AR activity in castrate resistant settings remain understudied. Here we show that protein phosphatase 1 regulatory subunits, identified from a phosphatase RNAi screen, direct PP1 catalytic subunits to a varied yet significant response in AR function. As such, we have characterised the PP1β holoenzyme, myosin phosphatase (MLCP), as a novel ligand independent regulator of the AR. Sustained MLCP activity through down-regulation of the MLCP inhibitory subunit, PPP1R14C, results in impaired AR nuclear translocation, protein stability and transcriptional activity in distinct models of PC progression, culminating in restoration of a non-malignant prostate genotype. Phenotypically, a marked reduction in cell proliferation and migration, characterised by G1 cell cycle arrest is observed, confirming PP1 holoenzyme disruption as a novel treatment approach in PC.

BACKGROUND: International consensus recognises four medulloblastoma molecular subgroups - WNT (MBWNT), SHH (MBSHH), Group 3 (MBGrp3) and Group 4 (MBGrp4) - each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. Subgroups harbor distinct clinico-pathological and molecular features, underpin current disease sub-classification and initial subgroup-directed therapies are underway in clinical trials (i.e. reduced risk-adapted treatments for favorable-risk MBWNT patients; SMO inhibitors for MBSHH patients). However, significant biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. METHODS: We undertook comprehensive molecular profiling and unsupervised class discovery (non-negative matrix factorization, t-SNE) of test and validation cohorts (n=704 in total), to identify consensus primary molecular subgroups within childhood medulloblastoma (3.0 years). FINDINGS: Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified, characterized by distinct biological/clinical features. For instance, MBSHH comprised two age-dependent subgroups, while MBGrp3 and MBGrp4 each split into two subgroups with significantly different survival rates. Survival analysis identified secondary features predictive of outcome. Cross-validated subgroup-dependent models incorporating these novel subgroups along with secondary features and established disease risk-factors, outperformed current disease risk-stratification schemes. These schema stratified patients into four clinical risk-groups - favorable-risk (91% 5-year survival, 25% of patients), standard-risk (81%, 23%), high-risk (42%, 38%) and very high-risk (28%, 13%) - to be considered for treatment reduction, intensification or novel therapies respectively. INTERPRETATION: The discovery of seven novel, clinically-significant, subgroups significantly improves disease risk-stratification and provides a new foundation for future research and clinical investigations.

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.

To identify potential regulators of propagation and self-renewal of Acute Lymphoblastic Leukaemia (ALL), we performed an explorative genome-wide RNAi screen followed by CRISPR ex vivo and in vivo validation screens in the t(4;11)-positive ALL cell line SEM. These screens identified the splicing factor PHF5A as a crucial component of the leukemic program. PHF5A is a subunit of the SF3b protein complex, which directs alternative splicing by binding to the branchpoint of pre-mRNA. Mutations in members of this complex including SF3B1 have been implicated in several haematological malignancies. Functional perturbation experiments demonstrated that PHF5A depletion impairs proliferation, viability and clonogenicity in a range of ALL and AML cell lines strongly suggesting that PHF5A is required for leukemic propagation and self-renewal. To identify genetic programs affected by PHF5A inhibition, we performed RNA-seq followed by analysis of differential gene expression and splicing events. We identified 473 genes with differential expression upon PHF5A knockdown. In addition, we performed in-depth analysis of splicing patterns by examining both differential exon/intron usage and exon junction formation. These analyses demonstrated that loss of PHF5A affects splicing of more than 2500 genes with exon skipping and intron retention being the most frequent splicing events. In order to identify processes and pathways affected by PHF5A, we performed gene set enrichment analysis (GSEA) on both differential expression and splicing. While gene sets associated with RNA processing including splicing, turnover and translation were enriched in both data sets, the differential gene expression signature was also linked to DNA repair processes including base excision, mismatch and homologous recombination repair. In line with these findings, knockdown of either PHF5A or its partner protein SF3B1 induced DNA strand breaks as indicated by comet assay and increased y-H2AX levels. Furthermore, both PHF5A and SF3B1 depletion sensitized ALL cells towards the DNA crosslinking agent mitomycin C. Closer inspection of RNA-seq datasets revealed reduced FANCD2 expression and skipping of exon 22 associated with impaired mono-ubiquitination of the FANCD2 protein as a consequence of PHF5A and SF3B1 knockdown. Furthermore, expression of RAD51, a key component of double strand break repair, also decreased upon PHF5A and SF3B1 knockdown. Notably, in vitro pharmacological inhibition of SF3b complex activity using H3B-8800 (or Pladienolide B) showed a very similar effect on FANCD2 expression, and ubiquitination as well as decrease of RAD51 and an increase in y-H2AX levels on a dose and time-dependent manner. This strongly suggests a mechanistic link between impaired RNA splicing and the repair of DNA double-strand breaks. These combined data show that leukemic cells are highly dependent on a functional SF3b splicing complex. Interference with its function results in DNA damage and also sensitizes towards DNA damaging agents pointing towards a possible benefit of the combined application of inhibitors targeting the SF3b complex with more conventional chemotherapy. Disclosures Ponthan: Epistem Ltd: Employment. Zwaan:Sanofi: Consultancy; Incyte: Consultancy; BMS: Research Funding; Roche: Consultancy; Janssen: Consultancy; Daiichi Sankyo: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Pfizer: Research Funding; Celgene: Consultancy, Research Funding. Vormoor:Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.