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Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations. Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters. Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse. Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.

Primary therapy resistance is a major problem in acute myeloid leukemia treatment. We set out to develop a powerful and robust predictor for therapy resistance for intensively treated adult patients. We used two large gene expression data sets (n=856) to develop a predictor of therapy resistance, which was validated in an independent cohort analyzed by RNA sequencing (n=250). In addition to gene expression markers, standard clinical and laboratory variables as well as the mutation status of 68 genes were considered during construction of the model. The final predictor (PS29MRC) consisted of 29 gene expression markers and a cytogenetic risk classification. A continuous predictor is calculated as a weighted linear sum of the individual variables. In addition, a cut off was defined to divide patients into a high-risk and a low-risk group for resistant disease. PS29MRC was highly significant in the validation set, both as a continuous score (OR=2.39, P=8.63·10−9, AUC=0.76) and as a dichotomous classifier (OR=8.03, P=4.29·10−9); accuracy was 77%. In multivariable models, only TP53 mutation, age and PS29MRC (continuous: OR=1.75, P=0.0011; dichotomous: OR=4.44, P=0.00021) were left as significant variables. PS29MRC dominated all models when compared with currently used predictors, and also predicted overall survival independently of established markers. When integrated into the European LeukemiaNet (ELN) 2017 genetic risk stratification, four groups (median survival of 8, 18, 41 months, and not reached) could be defined (P=4.01·10−10). PS29MRC will make it possible to design trials which stratify induction treatment according to the probability of response, and refines the ELN 2017 classification.

The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis. We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T. We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias. The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family. RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias. We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. Disclosures Browett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Background Acute Leukaemia (AL) is a genetically heterogeneous disorder caused by somatic mutations and acquired chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL/AF9 fusion, which results from the t(9;11)(p22;q23). A better understanding the molecular pathophysiology of AML, of the mechanisms of treatment resistance, disease relapse can be achieved by developing animal models. The MLL/AF9 fusion is frequently used to model AML in mice. However, to date, no MLL/AF9 leukemia models in zebrafish have been reported. Aim Our aim was to establish a transgenic zebrafish leukemia model using the human MLL/AF9 fusion gene. Methods To generate transgenic fish, two constructs (pTol2-Runx1+23: MLL-AF9-IRES-EGFP-cmlc-GFP and pTol2-Runx1+23: MLL-AF9-IRES-mCherry-cmlc-GFP) were injected together with Tol2 transposase mRNA into one-cell stage zebrafish embryos. We used the murine Runx1+23 enhancer to direct MLL/AF9 expression to hematopoietic stem cells and EGFP or mCherry as fluorescent markers. We selected transgenic embryos 24 hours post-fertilization based on the heart marker expression (cmlc). Results 29% (100 of 340 embryos) of the transgenics reached adulthood (6 weeks). After 6 to 24 months, 77% (77) of them developed signs of sickness. They became less active with protruding eyes and hump formation on the nose. Some started bleeding from the gills and/or showed tumor formation around the abdomen and head. Sick fish were euthanized and dissected. The autopsies showed pale and dysmorphic kidneys, pale and enlarged spleens, and in some cases white granular spots on the spleen. Histological sections revealed increased kidney, spleen, and liver cellularity with massive cellular infiltration of cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The transgenic F 0 zebrafish showed increased numbers of lymphoid cells (12 fish), precursor cells (9 fish), or myeloid cells (6 fish). Peripheral blood smears showed many intermediate-sized mononuclear cells with an increased nuclear-cytoplasmic ratio, with nuclei containing dipsersed chromatin and inconspicuous nucleoli resembling blasts. There were occasional myelocytes and no mature granulocytes. These data are consistent with the development of acute leukemia in our MLL/AF9 transgenic fish. Whole-mount in situ hybridization (WISH) was performed on F 1 embryos. RNA probes for early hematopoietic markers (gata1, scl, runx1, ikaros, cmyb, mpx, and lyz) were hybridized to 24 and 48 hpf F1 transgenic embryos. There were expression changes of these markers compared to age-matched wild-type larvae, including low expression of gata1, scl, cmyb and high expression of lyz, mpx and ikaros in the caudal hematopoietic organ. We also performed transplantation experiments with the kidney marrow cells from diseased fish to test whether the disease was transplantable. The disease was serially transplantable into secondary and tertiary recipient fish. Transplanted fish had a significantly shorter latency to disease development of only 2 to 6 weeks. The morphological evidence and the serial transplantability of the disease proves that we have in fact succeeded in establishing an MLL/AF9-driven acute leukemia model in zebrafish. The long latency and incomplete penetrance observed in our F 0 MA9 zebrafish, along with a shorter latency in the transplanted fish, suggests that additional somatic mutations are required for leukemogenesis in this model. We performed whole exome sequencing to find cooperating somatic mutations and RNASeq to identify differentially expressed genes in MA9 leukemia fish. Whole exome sequencing on six samples identified putative somatic mutations in genes such as Stat5, Cyp2j20, Ms4a17a.3, Tapbp.1 and Herc5.3, which have been reported to be mutated in human cancer. RNA-seq analysis on seven samples showed 67 differentially expressed genes with a q value < 0.05 (e.g., cxcl32b.1, myof1, ctdsp2, egr3, il2rb) and nine enriched pathways with a P-value of < 0.054 (e.g.: KRAS, TP53, MEK) in our transgenic leukemic MLL/AF9 fish. Conclusion Our MLL/AF9 zebrafish acute leukemia model will be a helpful tool to understand leukemia biology and enable testing of new therapeutic strategies. Disclosures Browett: AbbVie: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Myeloid leukemia is caused by acquired genetic changes in haematopoietic stem cells. The combination of stepwise acquisition of genetic changes together with selection of the fittest clones results in great genetic and clonal heterogeneity. We used a CALM-AF10-driven retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) to study clonal hierarchy and clonal evolution starting with a primary leukaemia (Fig 1: Leu7) which developed after 131 days and had B220 marker expression on 4% of its cells. Limiting dilution assays (LDAs) showed that the leukemia stem cell (LSC) frequency of Leu7 was 1:2339 (95% confidence interval: 1:794-1:6885). Whole exome sequencing (WES) and analysis of the variant allele fraction of somatic mutations revealed that Leu7 was composed of a main clone (Fig 1: grey) with two subclones (blue and red). Half a million leukemic cells from Leu7 were transplanted into 4 sublethally irradiated recipients, which all developed secondary leukemias after a latency of 19 days (Leu7Sec1 to 4). All secondary leukemias showed similar B220 expression levels to Leu7, and all showed an expansion of the blue subclone. When again half a million cells each of one of the secondary leukemias (Leu7Sec2) were transplanted into 4 recipients, the expansion of the blue subclone continued, the red subclone vanished and, surprisingly, the proportion of B220 expressing cells increased to between 16 to 26%. LDAs showed that the LSC frequency of Leu7Sec2 had not changed. However, several of the leukemias from the LDAs had greatly varying latencies (27 to 193 days) and B220 marker expression (2 to 85%). Four of these tertiary LDA leukemias (Leu7Sec2Ter5 to 8), which each arose from a single LSC, were analysed more closely using WES. Leu7Sec2Ter5 showed a similar latency (27 days) and B220 expression levels like Leu7SecTer1 to 4 and also had the expansion of the blue subclone. Leu7Sec2Ter6 had a long latency of 69 days and a very low B220 expression. Leu7Sec2Ter6 was driven by a new, third subclone (pink), and both the blue and the red subclone disappeared. Very interestingly, Leu7Sec2Ter7 and Leu7Sec2Ter8 had a very long latency of 193 days, and showed an expansion of a subclone (green) of the red subclone. The B220 expression was high (37%) to very high (85%) in these two leukemias. Taken together, these observations paint an interesting picture with the blue subclone outcompeting the red subclone, as leukemias arising from the red subclone only appear after a long latency and in leukemias initiated by a single LSC, when there is no blue subclone LSC present. As the four leukemias (Leu7Sec2Ter5 to 8), which each were derived from a single LSC, showed striking differences in latency and surface marker expression, it can be concluded that this variation in phenotype is an intrinsic property of an individual LSCs most likely a consequence of the distinct combination of somatic mutations present in the individual LSCs. These observations also suggest that distinct LSCs with different properties might be present in a single human leukemia. Figure 1 Figure 1. Disclosures Browett: Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

The discovery of CRISPR-Cas systems in prokaryotic adaptive immunity has provided a veritable cornucopia of molecular biology tools. Here, we review the remarkable adaptation of CRISPR-Cas ...

In adult acute myeloid leukemia (AML), the karyotype of the leukemic cell is among the strongest prognostic factors. The Medical Research Council (MRC) and the European LeukemiaNet (ELN) classifications distinguish between favorable, intermediate and adverse cytogenetic risk patients who differ in their treatment response and overall survival. Conventional cytogenetic analyses are a mandatory component of AML diagnostics but they are time-consuming; therefore, therapeutic decisions in elderly patients are often delayed. We investigated whether a screening approach using a panel of seven fluorescence in situ hybridization (FISH) probes would allow rapid identification of adverse chromosomal changes. In a cohort of 334 AML patients, our targeted FISH screening approach identified 80% of adverse risk AML patients with a specificity of 99%. Incorporating FISH screening into diagnostic workup has the potential to accelerate risk stratification and treatment selection, particularly in older patients. This approach may allow therapeutic decisions more quickly, which benefits both patients and physicians and might save costs.

Background With the emergence of immunotherapies as a promising cancer treatment, there is now a pressing need for pre-clinical animal models to test immunotherapy strategies for acute myeloid leukemia (AML). While murine xenotransplant models generated by transplanting human AML cells are frequently used to model AML, they require immune-deficient mice. Syngeneic murine bone marrow transplant leukemia models (MBMTLM), which are established in immune competent mice, usually require radiation of the recipient mouse before leukemic cells are transplanted. This radiation suppresses the immune system and makes it difficult to study how the immune system responds to leukemia cells. Aim Our aim was to establish immunocompetent MBMTLMs to understand how the immune system responds to leukemia cells expressing a highly immunogenic antigen. Method We first established MBMTLMs models driven by the CALM/AF10 or MLL/AF9 fusion genes. Then leukemia cells were transduced with a SIINFEKL expressing retrovirus (MSCV-DsRed-SIINFEKL). SIINFEKL is a highly immunogenic eight amino acid peptide from ovalbumin. Results The transduction efficiency of MSCV-DsRed-SIINFEKL was about 12.9% on leukemic cells of MLL/AF9 and 13% on CALM/AF10 cells. All primary MLL/AF9-SIINFEKL (n=3) developed leukemia with a latency of 22 days. SIINFEKL expression was detected on 75.5±3% of the spleen cells of these mice. These spleen cells were transplanted into irradiated (n=6) and non-irradiated (n=8) mice to establish secondary MLL/AF9-SIINFEKL leukemias (Table 1). In the irradiated recipients, five out of six mice developed leukemia within 22-29 days. In non-irradiated recipients, four out of eight developed leukemia with a latency of 29-45 days. Flow cytometry showed that SIINFEKL was expressed on 82.28±6% of the spleen cells in irradiated recipients. In contrast, fewer than 1% of the spleen cells in non-irradiated mice with secondary MLL/AF9-SIINFEKL leukemia expressed SIINFEKL. Similar experiments were performed with the CALM/AF10 model. All primary CALM/AF10-SIINFEKL transplanted mice (n=2) developed leukemia with a latency of 32 and 42 days and showed SIINFEKL positivity on 99% of the leukemia cells. Secondary CALM/AF10-SIINFEKL leukemias were generated by transplanting these AML cells into irradiated (n=6) and non-irradiated (n=8) mice. All six irradiated recipients developed leukemia within 21-40 days. However, only five of the eight non-irradiated recipients developed leukemia with a latency of 35-42 days. Strikingly, about 99% of the AML cells in the irradiated mice were SIINFEKL positive but fewer than 1% of the AML cells in the five non-irradiated mice who developed leukemia were SIINFEKL positive. Discussion We find that for both MLL/AF9 and CALM/AF10 AML an intact immune system (non-irradiated recipients) is able to largely eliminate AML cells which express the SIINFEKL antigen, even when challenged with a large number of AML cells. In the non-irradiated recipients only 50 to 60% developed leukemia, and the leukemias that did develop had hardly any SIINFEKL-positive cells. In contrast, nearly all irradiated mice (11 of 12) developed leukemia with a high percentage of SIINFEKL expressing AML cells. SIINFEKL was presented on the surface of leukemic cells by the murine MHC class I H-2Kb molecule. Our results differ slightly from research reported by Hasegawa et al. 2015, who showed that MLL/AF9 AML cells expressing ovalbumin caused leukemia after transplantation into non-irradiated mice. However, they did not investigate whether ovalbumin was still being expressed on the leukemia cells. Conclusion We have established syngeneic murine AML models in immunocompetent mice and have evidence that an intact immune system has the ability to suppress or even eliminate rapidly proliferating AML cells if they express a strong antigen. These models should be useful for developing immunotherapy strategies for AML. Disclosures Desai: University of Auckland: Current Employment. Kakadia:University of Auckland: Current Employment. Browett:University of Auckland: Current Employment; BeiGene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Shire: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bohlander:University of Auckland: Current Employment; Family of Marijana Kumerich: Research Funding; Leukaemia and Blood Cancer New Zealand: Research Funding.

The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.

There is experimental and observational evidence that the cells of the leukemic clone in acute myeloid leukemia (AML) have different phenotypes even though they share the same somatic mutations. The organization of the malignant clone in AML has many similarities to normal hematopoiesis, with leukemia stem cells (LSCs) that sustain leukemia and give rise to more differentiated cells. LSCs, similar to normal hematopoietic stem cells (HSCs), are those cells that are able to give rise to a new leukemic clone when transplanted into a recipient. The cell of origin of leukemia (COL) is defined as the normal cell that is able to transform into a leukemia cell. Current evidence suggests that the COL is distinct from the LSC. Here, we will review the current knowledge about LSCs and the COL in AML.

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are often ineffective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10-positive leukemias which may lay the foundation for novel targeted therapies. Here we demonstrate that the Polycomb Repressive Complex 1 gene BMI1 is consistently overexpressed in adult and pediatric CALM-AF10-positive leukemias. We demonstrate that genetic Bmi1 depletion abrogates CALM-AF10-mediated transformation of murine hematopoietic stem and progenitor cells (HSPCs). Furthermore, CALM-AF10-positive murine and human AML cells are sensitive to the small-molecule BMI1 inhibitor PTC-209 as well as to PTC-596, a compound in clinical development that has been shown to result in downstream degradation of BMI1 protein. PTC-596 significantly prolongs survival of mice injected with a human CALM-AF10 cell line in a xenograft assay. In summary, these results validate BMI1 as a bona fide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements.

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are often ineffective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10-positive leukemias which may lay the foundation for novel targeted therapies. Here we demonstrate that the Polycomb Repressive Complex 1 geneBMI1is consistently overexpressed in adult and pediatric CALM-AF10-positive leukemias. We demonstrate that geneticBmi1depletion abrogates CALM-AF10-mediated transformation of murine hematopoietic stem and progenitor cells (HSPCs). Furthermore, CALM-AF10-positive murine and human AML cells are profoundly sensitive to the small-molecule BMI1 inhibitor PTC209 as well as to PTC596, a compound in clinical development that has been shown to result in downstream degradation of BMI1 protein. PTC-596 significantly prolongs survival of mice injected with a human CALM-AF10 cell line in a xenograft assay. In summary, these results validate BMI1 as abonafidecandidate for therapeutic targeting in AML with CALM-AF10 rearrangements.

Calcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRLhigh AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR- Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML.

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P

// Isabella Barbutti 1 , Juliana M. Xavier-Ferrucio 1, * , Joao Agostinho Machado-Neto 1, # , Lauremilia Ricon 1 , Fabiola Traina 2 , Stefan K. Bohlander 3 , Sara Teresinha Olalla Saad 1 , Leticia Frohlich Archangelo 1, 4 1 Hematology and Hemotherapy Center, State University of Campinas (UNICAMP), Carlos Chagas 480, Campinas-SP, Brazil 2 Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil 3 Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand 4 Department of Cellular and Molecular Biology and Pathogenic Bioagents, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil * Present address: Department of Laboratory Medicine, Yale Stem Cell Center # Present address: Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil Correspondence to: Leticia Frohlich Archangelo, email: leticiafa@fmrp.usp.br Keywords: CATS (FAM64A), proliferation, clonogenicity, CALM/AF10, leukemogenesis Received: November 10, 2015 Accepted: August 21, 2016 Published: August 31, 2016 ABSTRACT The CATS (FAM64A) protein interacts with CALM (PICALM) and the leukemic fusion protein CALM/AF10. CATS is highly expressed in leukemia, lymphoma and tumor cell lines and its protein levels strongly correlates with cellular proliferation in both malignant and normal cells. In order to obtain further insight into CATS function we performed an extensive analysis of CATS expression during differentiation of leukemia cell lines. While CATS expression decreased during erythroid, megakaryocytic and monocytic differentiation, a markedly increase was observed in the ATRA induced granulocytic differentiation. Lentivirus mediated silencing of CATS in U937 cell line resulted in somewhat reduced proliferation, altered cell cycle progression and lower migratory ability in vitro; however was not sufficient to inhibit tumor growth in xenotransplant model. Of note, CATS knockdown resulted in reduced clonogenicity of CATS-silenced cells and reduced expression of the self-renewal gene, GLI-1 . Moreover, retroviral mediated overexpression of the murine Cats in primary bone marrow cells lead to decreased colony formation. Although our in vitro data suggests that CATS play a role in cellular processes important for tumorigenesis, such as cell cycle control and clonogenicity, these effects were not observed in vivo .

Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3. We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150–based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470–80. ©2016 AACR.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy., Mir-22 has been shown to be an oncogenic microRNA in breast cancer and myelodysplastic syndrome. Here, the authors show that mir-22 functions as a tumour suppressor in de novo acute myeloid leukaemia by inhibiting the expression of several oncogenes and that restoring mir-22 expression suppresses AML progression.

We report the case of a 26 year-old patient presenting with a persistent leukocytosis and CML-like marrow but no evidence of a BCR/ABL1 fusion. Molecular cytogenetics revealed that a portion of the ETV6 locus was inserted into the ABL1 locus. An ETV6/ABL1 fusion transcript could subsequently be confirmed. The patient was started on imatinib and went into complete cytomorphological remission. QRT-PCR measurements showed a 4log reduction of the ETV6/ABL1 fusion. 15 months later, the disease transformed into ALL and the patient expired. Thus, an ETV6/ABL1 fusion positive MPN has the potential to transform very rapidly into ALL.

Dose-dense induction with the S-HAM regimen was compared to standard double induction therapy in adult patients with newly diagnosed acute myeloid leukemia. Patients were centrally randomized (1:1) between S-HAM (2nd chemotherapy cycle starting on day 8 = “dose-dense”) and double induction with TAD-HAM or HAM(-HAM) (2nd cycle starting on day 21 = “standard”). 387 evaluable patients were randomly assigned to S-HAM (N = 203) and to standard double induction (N = 184). The primary endpoint overall response rate (ORR) consisting of complete remission (CR) and incomplete remission (CRi) was not significantly different (P = 0.202) between S-HAM (77%) and double induction (72%). The median overall survival was 35 months after S-HAM and 25 months after double induction (P = 0.323). Duration of critical leukopenia was significantly reduced after S-HAM (median 29 days) versus double induction (median 44 days)—P < 0.001. This translated into a significantly shortened duration of hospitalization after S-HAM (median 37 days) as compared to standard induction (median 49 days)—P < 0.001. In conclusion, dose-dense induction therapy with the S-HAM regimen shows favorable trends but no significant differences in ORR and OS compared to standard double induction. S-HAM significantly shortens critical leukopenia and the duration of hospitalization by 2 weeks.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line–derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Alterations of RUNX1 in acute myeloid leukemia (AML) are associated with either a more favorable outcome in the case of the RUNX1/RUNX1T1 fusion or unfavorable prognosis in the case of point mutations. In this project we aimed to identify genes responsible for the observed differences in outcome that are common to both RUNX1 alterations. Analyzing four AML gene expression data sets (n = 1514), a total of 80 patients with RUNX1/RUNX1T1 and 156 patients with point mutations in RUNX1 were compared. Using the statistical tool of mediation analysis we identified the genes CD109, HOPX, and KIAA0125 as candidates for mediator genes. In an analysis of an independent validation cohort, KIAA0125 again showed a significant influence with respect to the impact of the RUNX1/RUNX1T1 fusion. While there were no significant results for the other two genes in this smaller validation cohort, the observed relations linked with mediation effects (i.e., those between alterations, gene expression and survival) were almost without exception as strong as in the main analysis. Our analysis demonstrates that mediation analysis is a powerful tool in the identification of regulative networks in AML subgroups and could be further used to characterize the influence of genetic alterations.

Human megakaryocytes release glutamate and express glutamate-gated Ca 2 + -permeable N -methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca 2 + responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca 2 + flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca 2 + entry into Meg-01 cells, amplified Ca 2 + responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5–100 μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100 μM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC 50 20 μM and 36 μM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca 2 + entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205∗) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).

Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ≥1 mutation during remission at a VAF of ≥2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (p

Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR- ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P 0.001, 8/14, 57% vs. 26/111, 23%; P = 0.02). Furthermore, we identified DNMT3B to be rarely but recurrently targeted by truncating mutations in AML. Gene expression analysis of 13 patients with del(9q) and 454 patients with normal karyotype or various cytogenetic aberrations showed significant down regulation of TLE4 in patients with del(9q) (P = 0.02). Interestingly, downregulation of TLE4 was not limited to AML with del(9q), potentially representing a common mechanism in AML pathogenesis. Our comprehensive genetic analysis of the del(9q) subgroup reveals a unique mutational profile with the frequency of DNMT3A mutations in the del(9q) only subset being the highest reported so far in AML, indicating oncogenic cooperativity. © 2016 Wiley Periodicals, Inc.

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

Background:The small molecule BCL-2 inhibitor Venetoclax (VEN) has emerged as a promising frontline therapy in combination with low dose cytarabine (LDAC) or hypomethylating agents in older patients with acute myeloid leukemia (AML)(DiNardo et al, Lancet Oncol 2018, Blood 2019; Wei et al,JCO 2019). The anti-leukemic activity of single agent VEN as monotherapy in newly diagnosed AML is unknown. This information would be useful for studies considering VEN as a maintenance therapy. We have previously presented preliminary outcomes from a phase 1b/2 study: Chemotherapy and Venetoclax in Elderly AML Trial (CAVEAT), which incorporated a 7-day single agent VEN run-in phase prior to combination with chemotherapy (Wei et al, ASH 2018). The study procedures incorporated a bone marrow assessment performed at study screening and on day 8, prior to commencement of chemotherapy (Figure 1A). This allowed us to explore the single agent activity of venetoclax on bone marrow blasts in treatment naïve patients with AML and to assess molecular dynamics of response. Methods:The CAVEAT trial included 51 de novo or secondary patients with AML aged ≥65 years and considered fit for intensive chemotherapy. The study enrolled patients to 5 VEN dose cohorts (50-100-200-400-600mg). VEN was administered on days 1-14 during induction. Chemotherapy commenced on day 8, with a 7-day VEN monotherapy pre-phase. Molecular studies:a 54-gene myeloid NGS panel (Illumina TruSight) and MassARRAY MALDI-TOF mass spectrometry was performed on DNA extracted from bone marrow aspirates. FLT3-ITD testing was by fragment analysis, PCR and capillary electrophoresis. Minimal residual disease (MRD) testing was performed by digital droplet PCR for IDH mutations (assay sensitivity: 10-4-10-5). Results: A total of 46 patients had paired BM assessments at screening and on day 8 for comparison. A ≥50% relative reduction in BM blasts was recorded in 13/46 (28%) patients. Figure 1B shows the relative BM blast reduction stratified by molecular subgroups. Patients with NPM1(n=9) or IDH2 (n=8) mutation achieved greater BM blast reductions (median of 56% and 55%, respectively) than IDH1 (n=6), TP53 (n=10) or FLT3-ITD (n=5) mutant cases (median of 37%, 17% and 7%, respectively). Three NPM1 mutant cases with Clinically, complete remission (CR) and CR with incomplete count recovery (CRi) rates after chemotherapy were seen for the following mutations: NPM1(80%), IDH2 (100%), IDH1 (62%) and TP53 (36%) (Figure 1D). Despite all four response-evaluable patients with FLT3-ITD achieving CR/CRi, 3/4 relapsed with detectable FLT3-ITD (at 0.8, 1.5 and 12.3 months respectively). Median survival for each genomic sub-group were NPM1 (12.5m), IDH2 (not reached), IDH1 (6.1m),TP53 (3.8m), FLT3-ITD (5.5m). CyTOF analyses for changes in BCL-2 family expression are underway and results will be presented at the meeting. Conclusion: Treatment naïve NPM1 and IDH2 mutant AML blasts are highly sensitive to VEN alone and combination with cytarabine and anthracycline chemotherapy results in a high clinical response rate. TP53 and FLT3-ITD mutant cases were more resistant and outcomes were poor despite VEN combined with chemotherapy. Figure 1 Disclosures Reynolds: Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria. Fong:Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Astellas: Consultancy; Novartis: Speakers Bureau. Ting:NHMRC: Research Funding. Fleming:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Pfizer: Honoraria; Ariad: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Moujalled:Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Share in venetoclax royalties . Ivey:Novartis: Honoraria. Roberts:AbbVie: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Employment, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax. 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. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on correlative study results in the CAVEAT trial which combines venetoclax with modified intensive chemotherapy in AML, which is not an approved indication.

Background: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy and risk stratification based on genetic and clinical variables is standard practice. However, current models incorporating these factors accurately predict clinical outcomes for only 64-80% of patients and fail to provide clear treatment guidelines for patients with intermediate genetic risk. A plethora of prognostic gene expression signatures (PGES) have been proposed to improve outcome predictions but none of these have entered routine clinical practice and their role remains uncertain. Methods: To clarify clinical utility, we performed a systematic evaluation of eight highly-cited PGES i.e. Marcucci-7, Ng-17, Li-24, Herold-29, Eppert-LSCR-48, Metzeler-86, Eppert-HSCR-105, and Bullinger-133. We investigated their constituent genes, methodological frameworks and prognostic performance in four cohorts of non-FAB M3 AML patients (n= 1175). All patients received intensive anthracycline and cytarabine based chemotherapy and were part of studies conducted in the United States of America (TCGA), the Netherlands (HOVON) and Germany (AMLCG). Results: There was a minimal overlap of individual genes and component pathways between different PGES and their performance was inconsistent when applied across different patient cohorts. Concerningly, different PGES often assigned the same patient into opposing adverse- or favorable- risk groups (Figure 1A: Rand index analysis; RI=1 if all patients were assigned to equal risk groups and RI =0 if all patients were assigned to different risk groups). Differences in the underlying methodological framework of different PGES and the molecular heterogeneity between AMLs contributed to these low-fidelity risk assignments. However, all PGES consistently assigned a significant subset of patients into the same adverse- or favorable-risk groups (40%-70%; Figure 1B: Principal component analysis of the gene components from the eight tested PGES). These patients shared intrinsic and measurable transcriptome characteristics (Figure 1C: Hierarchical cluster analysis of the differentially expressed genes) and could be prospectively identified using a high-fidelity prediction algorithm (FPA). In the training set (i.e. from the HOVON), the FPA achieved an accuracy of ~80% (10-fold cross-validation) and an AUC of 0.79 (receiver-operating characteristics). High-fidelity patients were dichotomized into adverse- or favorable- risk groups with significant differences in overall survival (OS) by all eight PGES (Figure 1D) and low-fidelity patients by two of the eight PGES (Figure 1E). In the three independent test sets (i.e. form the TCGA and AMLCG), patients with predicted high-fidelity were consistently dichotomized into the same adverse- or favorable- risk groups with significant differences in OS by all eight PGES. However, in-line with our previous analysis, patients with predicted low-fidelity were dichotomized into opposing adverse- or favorable- risk groups by the eight tested PGES. Conclusion: With appropriate patient selection, existing PGES improve outcome predictions and could guide treatment recommendations for patients without accurate genetic risk predictions (~18-25%) and for those with intermediate genetic risk (~32-35%). Figure 1 Disclosures Hiddemann: Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Metzeler:Celgene: Honoraria, Research Funding; Otsuka: Honoraria; Daiichi Sankyo: Honoraria. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Beck:Gilead: Research Funding.

In chronic lymphocytic leukemia (CLL) patients, disruptions of the TP53 tumor suppressor pathway by 17p13 deletion (del17p), somatic TP53 mutations, or downregulation of microRNA-34a have been associated with a poor prognosis. So far, the impact of the various TP53 defects has not been evaluated in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53 mutations and 37 had del17p. In 24 (46%) of the TP53 mutated patients, no del17p was found and in 9 of the del17p patients, no TP53 mutation was identified. Based on a predicted proportion of TP53 disruption, a complete disruption of TP53 function, either by a combination of point mutations and/or del17p, was associated with a high risk for disease progression. Progression-free survival of patients with a heterozygous TP53 mutation was not significantly different from patients with a completely intact TP53 locus. In addition, only a complete loss of TP53 function correlated with low microRNA-34a expression levels. This trial was registered at www.clinicaltrials.gov as #NCT00090051.

Purpose To identify a robust prognostic gene expression signature as an independent predictor of survival of patients with acute myeloid leukemia (AML) and use it to improve established risk classification. Patients and Methods Four independent sets totaling 499 patients with AML carrying various cytogenetic and molecular abnormalities were used as training sets. Two independent patient sets composed of 825 patients were used as validation sets. Notably, patients from different sets were treated with different protocols, and their gene expression profiles were derived using different microarray platforms. Cox regression and Kaplan-Meier methods were used for survival analyses. Results A prognostic signature composed of 24 genes was derived from a meta-analysis of Cox regression values of each gene across the four training sets. In multivariable models, a higher sum value of the 24-gene signature was an independent predictor of shorter overall (OS) and event-free survival (EFS) in both training and validation sets (P < .01). Moreover, this signature could substantially improve the European LeukemiaNet (ELN) risk classification of AML, and patients in three new risk groups classified by the integrated risk classification showed significantly (P < .001) distinct OS and EFS. Conclusion Despite different treatment protocols applied to patients and use of different microarray platforms for expression profiling, a common prognostic gene signature was identified as an independent predictor of survival of patients with AML. The integrated risk classification incorporating this gene signature provides a better framework for risk stratification and outcome prediction than the ELN classification.

The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion., The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A, in these patients and show that the mutations reduce DNA binding activity.

Aldehyde dehydrogenase (ALDH) activity is an established feature of primitive normal human hematopoietic cells, in which it has been associated with a high expression of the 1A1 isoform of ALDH. High ALDH 1A1 activity has been reported to also characterize cells that propagate malignant populations arising in other tissues, but the regulation and basis of ALDH activity in primary human leukemic cells has not been well studied. We obtained samples from patients with newly diagnosed acute myeloid leukemia (AML; n = 21) and chronic myeloid leukemia (CML; n = 8) and analyzed different phenotypically and functionally defined subsets for their ALDH activity using the ALDEFLUOR ® kit and expression of the ALDH1A1 gene. We detected cells with high ALDH activity (ALDH pos ) in all samples from AML and CML patients. These were consistently enriched in the CD34 + population of these samples, but typically not in the CD34 + CD38 – subset. Leukemic cells with direct clonogenic activity in vitro or those able to repopulate the bone marrow of sublethally irradiated non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice were both ALDH pos and ALDH neg . Interestingly, ALDH1A1 transcripts were highest in the ALDH neg leukemic cells and, in studies with leukemic cell lines, exposure to an inhibitor of ALDH activity variably affected sensitivity to daunorubicin. Cells with high ALDH activity are commonly found within the CD34 + population of primary human leukemic cells but, unlike in normal hematopoietic tissues, do not selectively or consistently comprise those with proliferative potential or other distinct functional properties.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (33%) was limiting for CNA detection. We detected CNAs consistent with cytogenetics in 83% of AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc.

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder associated with bone fragility and susceptibility to fractures after minimal trauma. OI type V has an autosomal-dominant pattern of inheritance and is not caused by mutations in the type I collagen genes COL1A1 and COL1A2. The most remarkable and pathognomonic feature, observed in ∼65% of affected individuals, is a predisposition to develop hyperplastic callus after fractures or surgical interventions. To identify the molecular cause of OI type V, we performed whole-exome sequencing in a female with OI type V and her unaffected parents and searched for de novo mutations. We found a heterozygous de novo mutation in the 5′-untranslated region of IFITM5 (the gene encoding Interferon induced transmembrane protein 5), 14 bp upstream of the annotated translation initiation codon (c.−14C>T). Subsequently, we identified an identical heterozygous de novo mutation in a second individual with OI type V by Sanger sequencing, thereby confirming that this is the causal mutation for the phenotype. IFITM5 is a protein that is highly enriched in osteoblasts and has a putative function in bone formation and osteoblast maturation. The mutation c.−14C>T introduces an upstream start codon that is in frame with the reference open-reading frame of IFITM5 and is embedded into a stronger Kozak consensus sequence for translation initiation than the annotated start codon. In vitro, eukaryotic cells were able to recognize this start codon, and they used it instead of the reference translation initiation signal. This suggests that five amino acids (Met-Ala-Leu-Glu-Pro) are added to the N terminus and alter IFITM5 function in individuals with the mutation.

A nonrandom radial nuclear organization of genes has been well documented. This study provides further evidence that radial positioning depends on features of corresponding ∼1 Mbp chromatin domains (CDs), which represent the basic units of higher-order chromatin organization. We performed a quantitative three-dimensional analysis of the radial nuclear organization of three genes located on chromosome 1 in a DG75 Burkitt lymphoma-derived cell line. Quantitative real-time polymerase chain reaction revealed similar transcription levels for the three selected genes, whereas the total expression strength (TES) calculated as the sum of transcription of all genes annotated within a surrounding window of about 1 Mbp DNA differed for each region. Radial nuclear position of the studied CDs correlated with TES, i.e., the domain with the highest TES occupied the most interior position. Positions of CDs with stable TES values were stably maintained even under experimental conditions, resulting in genome-wide changes of the expression levels of many other genes. Our results strongly support the hypothesis that knowledge of the local chromatin environment is essential to predict the radial nuclear position of a gene.

Mutations of Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITDs) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in approximately 7% of AML patients. Patients carrying the FLT3-ITD but not the FLT3-TKD mutation have a significantly worse prognosis. Therefore, both FLT3 mutations seem to exert different biologic functions. FLT3-ITD but not FLT3-TKD has been shown to induce robust activation of the STAT5 signaling pathway. In the present study, we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD uses SRC to activate STAT5. Coimmunoprecipitation and pull-down experiments revealed an exclusive interaction between SRC but not other Src family kinases and FLT3-ITD, which is mediated by the SRC SH2 domain. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for SRC binding and subsequent STAT5 activation. Using site-specific Abs, we found that both residues were significantly more strongly phosphorylated in FLT3-ITD compared with FLT3-TKD. SRC inhibition and knock-down blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. We conclude that SRC might be a therapeutic target in FLT3-ITD+ AML.

Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.

A novel ABL1 fusion to the SH2 containing inositol phosphatase-1 ( SHIP1 ) in acute lymphoblastic leukemia (ALL)

Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing, using next generation technology, was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer, as well as acute myeloid leukemia (AML). Here, we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Mutations were detected by comparing the transcriptome sequence of an AML sample with the corresponding remission sample. Using this approach, we found five non-synonymous mutations specific to the tumor sample. They include a nonsense mutation affecting the RUNX1 gene, which is a known mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. Another missense mutation was identified in SHKBP1, which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in 95 cytogenetically normal AML patients was 2%. Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation.

Background: Venetoclax (VEN) is a potent small molecule BH3-mimetic drug that selectively targets the prosurvival protein BCL-2 and has an emerging role for treatment in Acute Myeloid Leukaemia (AML). This ongoing Phase 1b study aims to evaluate the optimal dose, safety and efficacy of VEN in combination with modified intensive chemotherapy in fit, elderly patients with AML who have not received prior induction chemotherapy. Here we present updated data including molecular correlates of response and treatment outcomes. Methods: Eligibility: Patients were enrolled with AML (excluding APL), age ≥65 years (≥60 years if monosomal karyotype), ECOG 0-1 and adequate organ function. Prior HMA/LDAC was permitted after a 14-day washout. Treatment: 1) Dose escalation cohorts comprised VEN 50mg (Cohort A), 100mg (B), 200mg (C), 400mg (D) or 600mg (E); 2) a 7-day VEN pre-phase incorporating dose ramp-up to minimise tumour lysis risk followed by a 7-day overlap with chemotherapy (day -6 to +7); 3) attenuated induction chemotherapy with cytarabine 100mg/m2/day IVI d1-5 staggered with idarubicin 12mg/m2 IV d2-3. For patients in remission, 4 cycles of consolidation were administered, comprising 14 days of VEN (day -7 to +7) with bolus cytarabine (100mg/m2/day IV d+1-2) and idarubicin (12mg/m2 IV d+1) followed by 7 cycles of VEN monotherapy maintenance; planned to commence q28d. Antifungal azoles were avoided during VEN exposure. The first patient was enrolled 17JUL2016. Molecular studies- Next generation sequencing using a 54-gene TruSight myeloid panel was performed on baseline bone marrow samples. FLT3-ITD testing was performed by capillary electrophoresis. RT-qPCR was used to detect minimal residual disease (MRD) in NPM1-mutated cases (assay sensitivity: 10-5-10-6). Results: The data cut-off date was 13JUL2018. 44 patients were enrolled with 3 patients still in the induction cycle. Median age was 72 years (range 63-80 years; 23% ≥75 years), 66% of patients were male, 41% had secondary AML and 38% prior hypomethylating agent (HMA) exposure. Main grade ≥3 non-haematological adverse events during induction were febrile neutropenia (56%), sepsis (32%), rapid atrial fibrillation (15%), diarrhoea (12%), nausea (10%) and localised infection (10%). No clinical TLS was observed. One hematologic dose-limiting toxicity was reported in cohort E (VEN 600mg), but a maximum tolerated dose has not been reached. We observed progressive delay in platelet count recovery as patients proceeded with each cycle of therapy (median time to platelets ≥50x109/L was 25d in induction, 37d in consolidation 1 and 57d in consolidation 4). Treatment-related mortality was 7% in induction. 18/38 (47%) had ≥30% relative reduction in bone marrow (BM) blasts after 7 days of pre-phase VEN monotherapy, with no correlation between VEN dosage and the degree of BM blast reduction. Overall CR/CRi rate was 71%. CR/CRi was achieved in 95% of patients with de novo AML (vs 42% in secondary/therapy-related AML). Response rates were 88% in intermediate vs 46% in adverse cytogenetic risk AML. Responses in patients with prior HMA therapy were 43% and 84% for previously untreated patients. Molecular data were available for 41 patients. 98% had ≥1 mutation detected. (Figure 1) Responses in response-evaluable patients were most common in NPM1 mutant AML (100%; n=7), followed by RUNX1 (90%; n=11), RAS (90%; n=10) and IDH (89%; n=9). Lowest responses were observed in TP53 mutant AML (33%; n=9). NPM1 MRD assessment was performed on 6 patients, of which 5 (83%) achieved NPM1 MRD negativity. (Figure 2) Updated response, response duration and survival data will be presented, along with molecular characteristics of relapse. Conclusion: To date, VEN up to 600mg in combination with 5+2 induction chemotherapy is tolerable in fit elderly patients with AML. Initial response rates >80% were observed in de novo AML, as well as NPM1, RUNX1, RAS and IDH mutant AML, whereas responses were lower in patients with prior HMA exposure, adverse karyotype, secondary and TP53 mutant AML. Molecular determinants of relapse require further study. Disclosures Wei: Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Amgen: Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Fong:Celgene: Other: Advisory board; Novartis: Other: Advisory board; Amgen: Research Funding; Pfizer: Other: Speaker fees. Reynolds:Novartis: Equity Ownership, Other: former employee of Novartis AG and holds stock in the company. . Roberts:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax.