ALBERT

Description: Introduction: While remission rates for childhood acute lymphoblastic leukemia (ALL) now exceed 80%, relapsed ALL remains the leading cause of non-traumatic death in children. Recently, a high-risk group of B-progenitor ALL patients has been identified. Such cases exhibit a gene expression profile similar to that of BCR-ABL1 positive (Ph+) ALL but are BCR-ABL1 negative, and also experience poor treatment outcomes. This subset, termed Ph-like ALL, is characterised by a range of genetic alterations that activate cytokine receptor and kinase signalling, allowing potential targeting by available tyrosine kinase inhibitors (TKI). The frequency of Ph-like ALL in the Australian community and the prognosis in the setting of the first MRD (minimal residual disease) intervention trial by the Australian and New Zealand Children's Haematology/Oncology Group (ANZCHOG ALL8) is unknown. Method: We retrospectively screened 250 unselected samples that were available from children diagnosed with B-ALL, for Ph-like ALL. The children, aged between 1 and 18 years, were enrolled on the ANZCHOG ALL8 trial and recruited from 2002-2011. The criteria for stratification to the high-risk group, based upon Berlin-Frankfurt-Munster (BFM) protocols, were BCR-ABL1 or MLL t(4;11) translocation; poor prednisolone response at day 8; failure to achieve remission by day 33 or high MRD (〉5 x10-4) at day 79. MRD was measured by RQ-PCR for patient-specific immunoglobulin and T-cell receptor rearrangements. All patients received a standard BFM four drug induction chemotherapy regimen including a prednisolone pre-phase and intrathecal methotrexate. High-risk patients received a further three novel intensive blocks of chemotherapy followed by transplant in most cases. Patients were screened for Ph-like ALL using a custom Taqman Low Density Array (TLDA) based upon previous reports. Fusions were then confirmed by RT-PCR for 30 known fusions, Sanger sequencing, mRNA sequencing and/or FISH. Results: Ten percent (25/250) of children in this cohort were identified as having Ph-like ALL, with most fusions converging on kinase activating pathways (Table 1). Three Ph-like ALL patients were considered high-risk, the remaining 22 (88%) were medium risk. Five children with Ph-like ALL, that did not have a fusion identified by RT-PCR, are currently under further investigation. Furthermore, 15 of the 20 (75%) of rearrangements involved CRLF2 with 10 (66%) of these children relapsing. Strikingly, 56% (14/25) of children in the ALL8 cohort who were identified as Ph-like subsequently relapsed compared to 16% (36/225) who were not, with significantly worse event free survival (p

Description: Eighty-six newly diagnosed Philadelphia-negative ALL pts were enrolled from 2012 to 2018, from 14 Australian centres; 82 pts were evaluable. Pts were stratified and treated as per the pediatric ANZCHOG Study 8 protocol based on BFM 2000. Response was assessed on day 33 and 79 by morphology, flow cytometry and RQ-PCR measurable residual disease (MRD) at a central lab according to EuroMRD criteria. Allogenic stem cell transplantation was permitted for high and very high-risk disease groups. Detailed genomic analysis was performed in 47 pts (to date), using whole transcriptome sequencing (mRNA Seq) and multiplex ligation-dependent probe amplification (MLPA) for recurrent ALL related gene deletions. Median age of the study was 24 (16 - 38) years; 28% were female; 59/82 (72%) had B-ALL. Median follow up was 36 (range 3-73) months. Induction mortality was 3.6%. CR rate at day 33 was 90.4% and day 79 (time point 2, TP2) 97.6%. Relapse free survival (RFS) at 2 years was 75.6% (95%CI 65.6 - 85.5%). CR rates at day 33 and day 79 were 90.4% and 97.6% respectively. The 2-year overall survival (OS) was 79.3% (18/82 events). In concordance with other studies, TP2 MRD predicted outcome in ALL06. MRD positive (pos) pts had a 2yr RFS of 68%, vs 98% in MRD negative (neg) pts (p=0.003). To date, 47 pts had mRNA Seq & MLPA; 11/47 pts had T cell ALL; 1/47 died during induction (2.1%). The median age of this subset was 21 (15-37) years, 23% were female and the RFS at 2 years was 73.97% (95%CI 65.6 - 91.44%). TP2 MRD remained predictive of outcome in this group with 2-year RFS in MRD pos pts 54% vs 95% in MRD neg pts (p=0.013, n=44). 13/47 pts have died with a 2-year OS of 73% (95%CI 62.7 - 90%). MPLA and mRNA Seq analysed independently of outcome data revealed 28/47 pts had genomic lesions categorise as High Risk (HR). These included fusions and structural genomic abnormalities involving KMT2A, IKZF1, IGH, ABL1, JAK, CRLF2, CDKN2A/B, PAX5, RAS and ZNF384. The remaining cases were classified as Standard Risk (SR) and included mainly hyperdiploid, T cell and ETV6-RUNX1 cases. Eleven of 13 pts who relapsed were genomic HR with poorer 2-RFS vs SR (59% vs 78.8%, p=0.023 respectively) (Fig 1.). We examined the relationship between genomics risk group and TP2 MRD, a known prognostic marker. Of the 22 pts who were MRD pos, 19 (86%) pts were in the HR genomics group. In contrast, for MRD neg pts, 13/22 were in the SR group (59%) (p=0.004 Fishers exact, Table 1). This demonstrates that the TP2 MRD positive group is strongly enriched for pts with HR genomics. Pts with HR genomics who were TP2 MRD pos had a 2 year RFS of 27% vs HR MRD neg or SR pts with a 2 yr RFS of 78% (p=0.001)(Fig. 2). Further, of the 13 deaths that were observed in this subset 9/13 (69%) fell within the group of pts with HR genomics/TP2 MRD+. The single induction mortality, for whom TP2 data was not available was also genomic HR. This is one of the first genomic surveys in a cohort of AYA pts, a group known for their inferior outcomes compared to children, treated on a pediatric inspired ALL protocol. Our overall outcomes compare favourably to other cohorts (EHA 2019 abstract 2416). In ALL06, genomic risk stratification based on previous published HR lesions, identified a HR cohort with significantly lower RFS and trend for inferior OS, vs a SR cohort. HR genomics was also associated with significantly higher rates of TP2 MRD positivity. Elucidation of targetable genomic lesions at the time of diagnosis may allow interventions to minimise MRD positivity and relapse in HR pts. Genomic information also improves understanding of underlying disease biology, providing targets for novel treatment discovery. Disclosures Yeung: Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Greenwood:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. White:AMGEN: Honoraria, Speakers Bureau; BMS: Honoraria, Research Funding.

Description: Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p=

Description: Abstract 413 Relapsed acute lymphoblastic leukemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. To identify novel sequence mutations contributing to relapsed in ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. The cohort included B-progenitor ALL with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), rearrangement of MLL (N=3), BCR-ABL1 (N=3), and low hyperdiploid, pseudodiploid, or miscellaneous karyotypes (N=10). This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including mutations in the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 63 diagnosis-relapse cases and 200 acute leukaemia cases that did not relapse found that 19% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis, acquired at relapse, or duplicated to homozygosity at the time of relapse. Moreover, several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations confer a selective advantage and promote resistance to therapy. The mutations either resulted in truncated alleles or deleterious substitutions in highly conserved residues of the HAT domain. To examine the functional consequences of the mutations, we introduced wild type or mutant Crebbp alleles into Cbp/Ep300flox/flox murine embryonic fibroblasts, (dKO MEFs), and examined histone acetylation, expression of CREBBP target genes, and cellular proliferation. The HAT domain mutations resulted in impaired acetylation of the key Crebbp substrate, H3K18, and resulted in impaired transcriptional regulation of multiple CREBBP targets and pathways, including cAMP, dsRNA and dexamethasone responsive genes. The latter observation suggests that CREBBP mutations may directly result in resistance to corticosteroid therapy, which is a hallmark of high risk ALL. Together, these data these results extend the landscape of genetic alterations in leukemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL. Disclosures: Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.

Description: Abstract 68 Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown. Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort. We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1). We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations. Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR. Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures: Evans: St. Jude Children's research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Treatment-resistant acute lymphoblastic leukemia (ALL) remains a significant clinical issue. Recently, genomic profiling has identified a new subtype of high-risk ALL termed Philadelphia-chromosome-like (Ph-like) ALL, associated with a poor outcome1. Ph-like ALL has a gene expression profile similar to Ph+ (BCR-ABL1+) ALL, characterized by the presence of fusion genes converging on kinase and cytokine signaling pathways. These pathways have been shown to be targetable both in vitro and in case reports by tyrosine kinase inhibitors (TKIs). Despite well-documented efficacy profiles, it is known from TKI-use in chronic myeloid leukemia (CML) andPh+ ALL that resistance is likely, resulting in relapse. Our study aims to model and understand mechanisms of TKI-resistance inPh-like ALL, informing future therapeutic strategies that may avert or overcome resistance, potentially improving patient outcomes. Methods Three Ph-like ALL lines were generated via retroviral-transduction from plasmids of fusion genes identified in patient cohorts (RANBP2-ABL1, SSBP2-CSF1R and PAX5-JAK2, a kind gift from C. Mullighan)2 into Ba/F3 pro-B cells. Transformation was confirmed via growth of cells in the absence of IL-3. Cells were tested for sensitivity to a panel of TKIs (imatinib, dasatinib, ponatinib, ruxolitinib and BMS-911543) via Annexin-V/7-AAD flow-cytometry and western blotting of downstream effector proteins. Drug resistance was generated through exposure of cells to incrementally increasing concentrations of TKIs over a period of 3-6 months, and cell death LD50 determined byAnnexin-V/7-AAD. Sanger sequencing of the 3-prime partner gene of each fusion was performed to identify the emergence of any kinase-domain mutations. Results Ba/F3 Ph-like cells demonstrated sensitivity to TKIs at clinically relevant doses (RANBP2-ABL1: 1 μM imatinib, 5 nM dasatinib & 5 nM ponatinib; SSBP2-CSF1R: 1 μM imatinib, 6 nM dasatinib; PAX5-JAK2: 1 μM ruxolitinib & 2 μM BMS-911543). This correlated with decreased levels of relevant downstream signaling proteins including p-Stat5, p-Erk and p-CrkL. TKI-resistant Ph-like ALL lines were tolerant to a significantly higher concentration of TKIs compared to control (RANBP2-ABL1: 10 μM imatinib, 200 nM dasatinib & 200 nM ponatinib; SSBP2-CSF1R: 10 μM imatinib, 200 nM dasatinib; PAX5-JAK2: 10μMruxolitinib & 10μM BMS-911543; Table 1). Sequencing analysis revealed that Ba/F3 RANBP2-ABL1 imatinib and dasatinib resistant cells acquired the clinically significant ABL1 T315I (c.944C〉T) kinase-domain mutation, which was ultimately targetable using the third-generation TKI ponatinib (LD50: 25 nM). An ABL1 E255K (c.763G〉A) and c-terminus deletion was discovered in the ponatinib-resistant line. In Ba/F3 SSBP2-CSF1R cells, a novel CSF1R L785M (c.2566C〉A) mutation was identified in imatinib-resistant cells whereas a deletion spanning the SSBP2-CSF1R breakpoint was acquired in the dasatinib-resistant line. A JAK2 Y931C (c.3286A〉G) point mutation previously associated with resistance to ATP-competitive inhibitors was acquired in Ba/F3 PAX5-JAK2ruxolitinib and BMS-911543 resistant lines. Conclusion In vitro modeling of Ph-like ALL resistance has identified novel kinase domain mutations and deletions that may arise as a result of targeted TKI therapy. In addition, previously identified mutations (T315I and E255K) were also identified. Detection of these mutations is important because alterations in drug-binding regions are known to result in significantly reduced TKI sensitivity, leading clinically to relapse3. This study describes an in vitro platform that can be utilized to inform future clinical approachesincluding the development of rational therapeutic approaches (and/or combination therapies) to avert resistance inPh-like ALL cases treated with rationally targeted therapies. Abbreviations: ABL1 - Abelson tyrosine protein kinase 1 CSF1R - Colony stimulating factor 1 receptor JAK2 - Janus kinase 2 PAX5 - Paired box 5 RANBP2 - RAN-binding protein 2 SSBP2 - Single-stranded DNA binding protein 2 References: 1 Den Boer et al, Lancet Oncology 2009; 10(2):125-34 2 Roberts et al, Cancer Cell 2012; 22(2):153-66 3 Barouch-Bentov & Sauer, Expert Opinion on Investigational Drugs 2011; 20(2);153-208 Disclosures Hughes: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.

Description: Mannose-binding lectin (MBL) is a mediator of innate immunity that influences the risk of infection in a range of clinical settings. We previously reported associations between MBL2 genotype and infection in a retrospective study of myeloablative allogeneic hematopoietic stem cell transplantation (allo-HCT). However, other studies have been inconclusive, and the role of MBL in reduced-intensity conditioning (RIC) transplantation is unknown. Here we report a prospective study examining MBL2 genotype, MBL levels, and risk of major infection following HLA-matched sibling myeloablative (n = 83) and RIC (n = 59) HCT. Baseline MBL levels were higher in recipients than donors (P 〈 .001), and recipient MBL levels increased during the peritransplantation period (P = .001), most notably in MBL2 wild-type individuals receiving myeloablative total body irradiation (mTBI). MBL2 coding mutations were associated with major infection in recipients receiving mTBI. The cumulative incidence of major infection in recipient harboring an MBL2 mutation receiving mTBI was 70.6%, compared with 31.1% of those without mutations not receiving mTBI (P = .01). MBL status was not associated with infection in RIC transplants. These results confirm the association of MBL status with risk of infection in myeloablative, TBI-conditioned transplantation. Studies examining the role of MBL replacement therapy to prevent infection in this setting should be considered.

Description: Introduction Down Syndrome (DS) patients are at high risk of developing hematological malignancies and ~10% are born with a pre-leukemic disorder characterised by the overproduction of immature megakaryoblasts. Children with DS have a 20-fold increased risk of developing acute lymphoblastic leukemia (ALL) of which 60% are associated with high expression of cytokine receptor like factor 2 (CRLF2) and of these, ~9% acquire the aggressive CRLF2 p.F232C mutation. DS-ALL children also experience high treatment toxicity and high relapse rates compared to non-DS leukemia patients. Genes on chromosome 21 including the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) are likely to play a role in DS leukemogenesis and may be targets for a personalized treatment approach. We aimed to determine if HMGN1 is necessary for leukemic cell proliferation using an inducible CRISPR/Cas9 guide (g)RNA murine xenograft model. Methods A DS leukemic cell line model was created using the human trisomy 21 megakaryoblastic SET-2 cell line harboring JAK2 p.V617F; the only trisomy 21 leukemic cell line currently available. SET-2 cells were transduced with CRLF2 p.F232C to model an aggressive DS-ALL mutation. NOD.Cg-Prkdcscid,Il2rgtm1Wjl/Szj (NSG) mice were each injected with 3x105 SET-2 CRLF2 p.F232C CRISPR/Cas9 cells expressing luciferase in 3 groups; Cas9 only control, HMGN1 gRNA, or JAK2 positive control gRNA. Doxycycline was administered post leukemic engraftment to induce the gRNAs and create a knockout (KO) and leukemic burden was monitored by bioluminescent imaging (BLI) twice weekly for the remainder of the experiment. Once Cas9 control mice became moribund, they were culled along with 50% of the JAK2 or HMGN1 KO mice and complete blood counts were performed. Bone marrow (BM), spleen and liver sections were stained with hematoxylin and eosin (H&E) and survival analysis was carried out for remaining JAK2 or HMGN1 KO mice. RQ-PCR was used to detect HMGN1 expression levels in KO mice organs at endpoint and DNA was extracted from cells harvested from each organ to undertake a gene editing analysis. Results Leukemic engraftment in mouse BM was observed 10 days post transplant with a radiance signal of ~1 x 104p/s/cm2/sr, therefore gRNAs were induced on day 11. On day 20, a significant reduction in tumor burden was detected in JAK2 and HMGN1 KO mice compared to Cas9 control mice (Fig. 1, Cas9: 8.4x105±1.7x105; JAK2 KO: 2.7x104±8.9x103; HMGN1 KO: 1.5x105±1.7x104 p/s/cm2/sr, prone: p

Description: Background and Objective 15% of pediatric and 40% of adult T-cell Acute Lymphoblastic Leukemia (T-ALL) patients fail conventional therapy highlighting the need for novel therapeutic strategies based on genomic alterations of individuals. We interrogated genomic alterations in Australian T-ALL patients for patterns of mutation and druggable targets. A subset of samples were used to establish patient-derived xenograft (PDX) models to evaluate novel therapies. Methods T-ALL patients' samples underwent next generation genomic analyses (128 total samples including diagnosis, refractory and relapse timepoints from 118 patients of all age groups). mRNA sequencing (mRNAseq) identified gene fusions and structural variants and assessed gene expression (n=101 patients). Fusions were called when identified by 2/3 predictors (FusionCatcher, SOAPfuse, JAFFA). Variant calling utilized GATK HaplotypeCaller and underwent several filtering steps to eliminate possible germline alterations and common SNPs. DNA copy number variations (CNVs) were detected via Multiplex Ligation-dependent Probe Amplification (MLPA: P202, P335, P383; n=64 patients). Establishment of PDX models from patient material (bone marrow or peripheral blood) is ongoing. Recapitulation of human disease was confirmed by mRNAseq in a subset of xenografts. Results Genomic fusion genes were identified in 46/101 samples (46%) by mRNAseq; the most common fusion identified was STIL-TAL1 (n=6). Increased expression of LCK and/or LAT (encoded proteins are involved in T-cell receptor (TCR) signal transduction) was observed in 100% of patients with the STIL-TAL1 gene fusion indicating TCR signaling pathways may be perturbed in this sub-group. Other common gene fusions were MLLT10-DDX3X (n=5) and KMT2A-MLLT4/AFDN (n=4). We also observed the previously reported fusions SET-NUP214, KMT2A-MLLT1, PICALM-MLLT10, NUP214-ABL1, several fusions involving TCR subunits as well as novel fusions involving KMT2A, NOTCH1, LMO1, ZEB2. Numerous nonsynonymous mutations were identified in 81/101 patients (80%) with mRNAseq data available (Figure 1). Broadly, the mutated genes encode proteins in the following categories: oncoproteins (NRAS, KRAS); tumour suppressors (TP53, CHEK2, BRCA1, BRCA2, PTEN), epigenetic regulators (EZH2, SETD5, DNMT3A); regulators of NOTCH signalling (NOTCH1, NOTCH2, FBXW7); transcription factors and regulators (IKZF1, KMT2A, EP400, SMARCD1, RUNX1, AFF1, AFF3); kinase and cytokine signal regulators (ATM, JAK2, JAK3, TYK2, FLT3, PTPN11). INDELS in clinically relevant genes were identified in 37/101 patients (37%) including alterations to: NOTCH1, PHF6, PTEN, STAT1, IL7R, CDKN2A, LYL1, WT1, JAK3, LEF1. The most common copy number alterations identified in our patient cohort were CDKN2A/B deletions (30/64 patients, 47%), PHF6 duplication (20/64 patients, 31%) and MLLT3 deletion (13/64 patients, 20%). In patients with CDKN2A/B deletions and additional CNVs, PHF6 duplication (n=6) and MYB duplication (n=4) were mutually exclusive. However, one patient without CDKN2A/B deletions harbored both MYB and PHF6 duplications. MLLT3 deletion always co-occurred with CDKN2A/B deletions (13/13 patients with MLLT3 deletion), but was never observed with PTEN deletion (0/7 patients with PTEN deletion). Patients with either CDKN2A/B deletions or PHF6 duplications frequently harbored NOTCH1 abnormalities: 13/32 patients (41%) and 8/21 patients (38%), respectively. PDX primagrafts investigated the engraftment latency and peripheral organ infiltration. Primagrafts established from patients harboring NUP214-SET1 or NUP214-ABL1 fusions engrafted at a slower rate (82 d and 91 d, respectively) than primagrafts from patients harboring a STIL-TAL1 fusion (45 d) or CDKN2A/B deletions (31 d and 48 d). Conclusions In our T-ALL cohort we demonstrate that the majority of cases harbor rearrangements, structural variations and duplication/deletion of genes associated with malignant transformation. We identified several co-occurring lesions as well as mutually exclusive genomic abnormalities. The top 20 mutated genes in our patient cohort differ to those reported for a pediatric cohort (Roberts et al 2019 Blood 134:649), indicating an association between patient age and genomic alteration. Secondary PDX models investigating novel targeted treatment strategies are ongoing. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.