MLL Partial Tandem Duplication Research Articles

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p<0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p<0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p<0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Molecular Pathogenesis of Progressive Refractoriness in MLL -Rearranged AML

BackgroundDespite advanced therapeutics, many leukemia patients become refractory to additional therapy, accounting for a major cause of leukemic deaths. Among major human acute myeloid leukemias (AMLs), MLL -rearranged AML is characteristic of progressive refractoriness to chemotherapy and shorter period to relapse. To clarify the molecular pathogenesis of such characteristics, we analyzed genetic abnormalities accumulated in MLL -rearranged AML in additional to the genetic events like MLL -fusion and MLL -partial tandem duplication (MLL-PTD).MethodWe exploited a mouse model of recurrent leukemia generated by mouse progenitors transduced with human MLL / AF9. Mouse leukemic cells with MLL / AF9 were serially transplanted in a total of 58 mice in 4 generations. We investigated somatic genetic events in leukemic cells from mouse models of MLL -rearranged AML and patients with MLL -rearranged AML. Whole exome and targeted-capture sequencing results were combined and analyzed to detect somatic genetic events and whole transcriptome sequencing and microarray dataset to assess gene expression.ResultsIn two independent experiments using mouse with transduced MLL/AF9, leukemic cells obtained in the 1st (N=2) and 4th (N=9) transplants were analyzed by whole exome sequencing. The onset of leukemia became progressively earlier in later generation transplant. Median periods to relapse were 57 and 16 days in the 1st and the 4th transplants, respectively (P < 0.001). With regard to molecular pathogenesis, leukemic cells in the 4th transplants had 12−31 more mutations, in addition to the mutations found in those in the 1st transplants (6-44 mutations). Significantly, two RNA polymerases (Polr2a and Polr3d), which are functionally related to Mll, were mutated in two experiments during the 4th transplants, suggesting that genetic lesions in MLL-associated complex could play a synergetic role in survival advantage of MLL/AF9 positive cells.In addition, mutations in Ptpn11 (G60R) and Gnb2 (G77R), which correspond to the same residues in human proteins, were also detected. In the cohort of human hematological neoplasms analyzed by targeted-capture sequencing, PTPN11 (G60R) is located at the very well documented hotspot in MLL -rearranged human AML and GNB2 G77 residue is present close to that affected by other recurrent mutations in human AML. Of note is that a germline mutation of the identical position of orthologous protein GNB1 was most recently reported in a case with acute lymphoblastic leukemia. To confirm pathological significance of such a novel GNB2 mutation, we analyzed gene expression in human AML. When compared between human MLL -rearranged (N=176) and MLL -rearrangement (−) (N=692) AML cases, GNB2 transcripts were significantly upregulated in human MLL -rearranged AML cases. Functionally, we induced wild-type and G77R mutant GNB2 in human hematopoietic cell lines. Overexpression of wild-type GNB2 conferred cytokine independency to granulocyte macrophage colony-stimulating factor dependent leukemic cell line TF1. Induced GNB2 (G77R)and knocked down wild-type GNB2 in MLL -rearranged AML cell lines were used to assess and confirm the molecular pathogenesis associated with a novel candidate protein GNB2.ConclusionsOur results in serial transplant experiments provide insights into the molecular mechanism of progression and progressive refractoriness of human MLL -rearranged AML. This study confirms that GNB2 is a novel candidate protein whose overexpression and activated mutation might play a significant role in the pathogenesis of this leukemia. DisclosuresTakaori-Kondo:celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria. Makishima:Yasuda Medical Foundation: Research Funding.

PS1040 DETECTION OF MRD MAY PREDICT THE OUTCOME OF PATIENTS WITH ACUTE MYELOID LEUKEMIA WITH BIALLELIC CEBPA MUTATIONS

Background: The somatic mutations that drive acute myeloid leukaemia (AML) are highly heterogenous. Identifying the mutations that drive individual cases can help determine patient prognosis and therapy. For this reason, genetic testing for prognostically important mutations in leukaemic DNA is now routine in many diagnostic laboratories. Also, the analysis of gene-expression profiles from AML RNA can provide additional clinically useful information that cannot be inferred from DNA sequencing. Whilst it is both expensive and impractical to carry out both types of sequencing and analyses, we hypothesised that the two could be combined more cheaply and effectively if the presence of mutations in prognostically important gene mutations could be identified from RNA-seq data. However, computational methodologies for robust detection of the diverse types of somatic mutations found in AML such as substitutions, indels, tandem duplications and translocations, are not currently available. Aims: To develop an stand-alone, lightweight and use-friendly software for the identification of clinically relevant mutations from AML RNA-seq data. Methods: To ensure efficient mapping of RNA-seq reads, we hash-indexed the DNA sequences of target genes using 10-mer sliding windows and implemented the ”seed and extend“ algorithm for read alignment. Point mutations and small indels were detected from reads with imperfect alignments and tandem duplications were detected from reads spanning the duplication junction. Translocations were detected from reads whose ends belonged to preselected fusion partner genes. Results: To benchmark our software we used RNA-seq data from 151 whole-exome/genome-sequenced AML samples studied by The Cancer Genome Atlas Research Network. We show that our software reliably calls clinically important mutations affecting the genes NPM1 (4-nt insertion), FLT3 (substitutions and internal tandem duplications, ITD), MLL partial tandem duplications (PTD), as well as substitutions in CEBPA, IDH1/2, TP53 and RUNX1. Furthermore, we identified gene fusions including PML-RARA, MYH11-CBFB, RUNX1-RUNX1T1, BCR-ABL1 and NUP98-NSD1. Our software is fast and memory efficient and is able to identify the above mutations in less than 20 minutes starting with RNA-seq FastQ files of 100 million 50 bp paired-end reads, using a standard modern laptop computer. In addition, the software operates through a graphical user interface making it accessible to users without programming knowledge. Summary/Conclusion: We demonstrated that clinically important somatic mutations that drives AML can be reliably detected from RNA-seq data alone using our software. As our approach can be readily combined with conventional gene expression analyses of the same RNA-seq dataset, it can be used to generate data with enhanced clinical utility that can improve prognostication and guide patient treatment. As RNA sequencing is a straightforward procedure, our approach can readily enter clinical laboratories, where it can significantly reduce experimental costs and accelerate diagnostic work-ups.

PS1039 ROBUST IDENTIFICATION OF CLINICALLY RELEVANT MUTATIONS IN AML USING RNA‐SEQ

Background:The somatic mutations that drive acute myeloid leukaemia (AML) are highly heterogenous. Identifying the mutations that drive individual cases can help determine patient prognosis and therapy. For this reason, genetic testing for prognostically important mutations in leukaemic DNA is now routine in many diagnostic laboratories. Also, the analysis of gene‐expression profiles from AML RNA can provide additional clinically useful information that cannot be inferred from DNA sequencing. Whilst it is both expensive and impractical to carry out both types of sequencing and analyses, we hypothesised that the two could be combined more cheaply and effectively if the presence of mutations in prognostically important gene mutations could be identified from RNA‐seq data. However, computational methodologies for robust detection of the diverse types of somatic mutations found in AML such as substitutions, indels, tandem duplications and translocations, are not currently available.Aims:To develop an stand‐alone, lightweight and use‐friendly software for the identification of clinically relevant mutations from AML RNA‐seq data.Methods:To ensure efficient mapping of RNA‐seq reads, we hash‐indexed the DNA sequences of target genes using 10‐mer sliding windows and implemented the ”seed and extend“ algorithm for read alignment. Point mutations and small indels were detected from reads with imperfect alignments and tandem duplications were detected from reads spanning the duplication junction. Translocations were detected from reads whose ends belonged to preselected fusion partner genes.Results:To benchmark our software we used RNA‐seq data from 151 whole‐exome/genome‐sequenced AML samples studied by The Cancer Genome Atlas Research Network. We show that our software reliably calls clinically important mutations affecting the genes NPM1 (4‐nt insertion), FLT3 (substitutions and internal tandem duplications, ITD), MLL partial tandem duplications (PTD), as well as substitutions in CEBPA, IDH1/2, TP53 and RUNX1. Furthermore, we identified gene fusions including PML‐RARA, MYH11‐CBFB, RUNX1‐RUNX1T1, BCR‐ABL1 and NUP98‐NSD1. Our software is fast and memory efficient and is able to identify the above mutations in less than 20 minutes starting with RNA‐seq FastQ files of 100 million 50 bp paired‐end reads, using a standard modern laptop computer. In addition, the software operates through a graphical user interface making it accessible to users without programming knowledge.Summary/Conclusion:We demonstrated that clinically important somatic mutations that drives AML can be reliably detected from RNA‐seq data alone using our software. As our approach can be readily combined with conventional gene expression analyses of the same RNA‐seq dataset, it can be used to generate data with enhanced clinical utility that can improve prognostication and guide patient treatment. As RNA sequencing is a straightforward procedure, our approach can readily enter clinical laboratories, where it can significantly reduce experimental costs and accelerate diagnostic work‐ups.

Mll-Partial Tandem Duplication (Mll-PTD) Causes Pseudohypoxia and Hypermethylation of the Epigenome through Suppression of Mitochondrial Complex II

The MLL-partial tandem duplication (MLL-PTD),characterized by the internal duplication of exons 3-9 or 3-11 in the MLL gene, produces an elongated protein, and is considered as a gain-of-function mutation. The MLL-PTD is primarily found in elderly patients with myelodysplastic syndromes and acute myeloid leukemiaas well as healthy individuals.Previously we showed that Mll-PTD knock-in (MllPTD/WT) mice presented enhanced self-renewal of hematopoietic stem cells (HSCs) and partially blocked differentiation of hematopoietic stem/progenitor cells (HSPCs). Interestingly, Mll-PTD increased the protein level of HIF1A in HSPCs, which is critical for enhanced self-renewal of HSCs. In the current study, we investigated the mechanisms for HIF1A activation by Mll-PTD.In normoxia, HIF1A is hydroxylated by prolyl hydroxylases (PHD), resulting in rapid protein degradation via ubiquitination. PHD is one of the well-known enzymes whose activity is dependent on the cellular level of α-ketoglutarate (α-KG), one of the metabolites in the tricarboxylic acid (TCA) cycle. Accumulation of subsequent metabolites of α-KG, such as succinate, fumarate, and malate, inhibits activity of α-KG-dependent enzymes. Indeed, mitochondrial dysfunction is known to result in accumulation of TCA cycle intermediates, leading to activation of HIF signaling. Thus, we first examined if Mll-PTD induces the alteration of mitochondrial functions. Interestingly, cellular respiration and activity of mitochondrial complexes (I, II, and III) were significantly decreased in HSPCs of MllPTD/WT mice, while the copy number of mitochondrial DNA was not altered. These results indicate that suppression of mitochondrial activity is not due to the decrease of the total mitochondria. We also examined mRNA expression levels of several major TCA cycle enzymes, and found that succinate dehydrogenase (Sdh) complex (Sdha, Sdhb, and Sdhd) was significantly downregulated in MllPTD/WT HSPCs. SDH is a critical TCA cycle enzyme which converts succinate to fumarate. Inactivation of SDH is known to result in impairment of mitochondrial biogenesis, a blockade of the TCA cycle, and accumulation of TCA cycle metabolites.We next quantified metabolites in glycolysis and TCA cycle in the plasma from WT control and MllPTD/WT mice. NMR analysis revealed that succinate, fumarate, and malate were increased in the plasma of MllPTD/WT mice. Especially, the ratios of fumarate and malate to α-KG were both significantly increased in MllPTD/WT compared to WT control. Indeed, post-α-KG metabolites increased HIF1A protein in human cord blood CD34+cells in vitro, indicating that higher levels of succinate, fumarate, and malate to α-KG levels stabilize HIF1A. We also confirmed that knockdown of Sdh increased the HIF1A protein level in murine cell line in normoxia. These results indicate that downregulation of Sdh in MllPTD/WT is one of the mechanisms for suppression of mitochondrial activity, leading to pseudohypoxia and HIF1A activation.Besides PHD, TET and histone lysine demethylases are also α-KG-dependent enzymes. We found that in MllPTD/WT HSPCs, the 5-methylcitosine (5-mC) level was increased in genomic DNA, and trimethylation levels at H3K4, H3K9, H3K36 and H3K79 were also increased. Collectively, these results suggest that metabolic pseudohypoxia due to lower mitochondrial activity not only activates HIF1A signaling but also induces hypermethylation in DNA and histones, through suppression of α-KG-dependent PHD and demethylases.In summary, we demonstrate that through suppression of mitochondrial complex II, Mll-PTD causes pseudohypoxia and hypermethylation of the epigenome, which may contribute to expansion of premalignant clones and accumulation of additional mutations in those cells. Interestingly, it has been proposed that IDH mutations are involved in tumorigenesis in leukemias and brain tumors through a similar mechanism. Moreover, loss-of-function mutations of the TCA cycle enzymes, SDH complex, and fumarate hydratase, are frequently found in various solid tumors associated with pseudohypoxia and hypermethylation phenotypes. Further investigations of the impact of metabolic-rewiring-mediated pseudohypoxia/hypermethylation on tumorigenesis may lead to the development of novel therapeutic strategies to prevent the onset and/or the progression of various types of malignant diseases. DisclosuresNo relevant conflicts of interest to declare.

Inducible Correction of a RUNX1 Mutation of Human AML Causes a Switch of AML to B-ALL

Background: Point mutations of RUNX1 gene are frequently seen in myeloid malignancies such as myelodysplasia (MDS) and acute myeloid leukemia (AML), and are associated with unfavorable clinical outcomes. AML patients bearing RUNX1 mutations have a significantly lower complete remission (CR) rate compared with patients with wild-type RUNX1 (30% vs. 73%), whereas in contrast, the CR rate of ALL patients, in which RUNX1 is rarely mutated, are at 92%. It remains unclear whether RUNX1 mutations found in AML patients are causal to AML maintenance, and if RUNX1 mutations serve as a valid therapeutic target in the treatment of such therapy-resistant AML.Experimental Approach: The RUNX1 mutations are found frequently associated with MLL-partial tandem duplication (MLL-PTD) in AML. We have developed two complementary mouse models that express a MLL-PTDknock-in mutation together with a RUNX1 S291fsX300 mutation found in AML patients: in the first model RUNX1 S291fsX300 was introduced into the bone marrow cells of the MLL-PTD knock-in mice by retrovirus transduction, while in the second model the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the Collagen a1 locus and the mice was crossed to MLL-PTD knock-in. In the second model, the mutant RUNX1 protein is only expressed when the mice were fed with doxycycline containing food and the RUNX1 mutant is absent or “corrected” upon doxycycline withdrawn. BM cells of these mice, as well as WT and MLL-PTD controls, were transplanted to syngeneic mice and the recipients were tracked for disease development and progression at bi-monthly intervals. The doxycycline induction or withdraw was carried out after secondary transplant, and the expression of RUNX1 S291fsX300 was monitored by a built-in GFP reporter and verified by RT-PCR.Under tetracycline induction, the RUNX1 S291fsX300 and MLL-PTD double mutation bearing mice developed a spontaneously AML and had a survival time 6-10 months after transplantation, and they showed symptoms of MDS/AML or AML, including thrombocytopenia, anemia, leukocytosis, splenomegaly, abnormal BM and spleen cell morphologies. We transplanted the AML mouse bone marrow to secondary recipients, and observed them with or without continuing doxycycline induction. By using the O-propargyl-puromycin to track the newly synthesized proteins, we found that the RUNX1 mut-on and RUNX1 mut-off mice showed comparable protein synthesis rate at full-blown leukemia stage, indicating that the RUNX1 mutation does not play a restrictive role to protein synthesis in transformed leukemia cells. The c-Kit+ AML cells cultured from the RUNX1 mut-off mice showed reduced viability and significantly reduced CFU-C activities, which are accompanied by a lineage switch in a decrease in expression of Mac1 and an increase in expression of B220. In vivo, the RUNX1 mut-off mice showed a slightly prolonged survival compared with RUNX1 mut-on group (182 vs. 126 days, p=0.0036). An analysis of the lineage distribution in their peripheral blood and bone marrow revealed that the RUNX1 mut-off AML mice underwent a dramatic lineage switch from myelocytes to lymphocytes (Fig. 1): while the Mac1+Gr1+population remained high in the RUNX1 mut-on AML mice, turning off RUNX1 mut expression caused an increased B cell population in expense of the Mac1+Gr1+population. Pathological analysis also showed a drastic decrease of the myeloid marker MPO and increased lymphoid marker B220 in the RUNX1mu-off mice. Consistently, a RT-PCR test of the BM c-Kit+ cells further showed that upon turning off the RUNX1mut, critical B cell regulatory genes including TCF3, EBF1, PAX5, CD79A were significantly up regulated whereas myeloid regulatory genes such as PU.1 were downregulated. In addition, RUNX1 S291fsX300 expression in the AML cells confers a resistance to chemotherapy (Ara-C) treatment compared with that of WT RUNX1 expression, indicating that the RUNX1 mutation is responsible for the AML resistance to therapy.Conclusion:Although RUNX1 mutations found in AML patient may not be essential for the hyperproliferative and growth phenotype of the AML, they play a causal role in maintaining myeloid lineage restriction, expansion and chemo-resistance. Correction or removal of the RUNX1 mutation in AML cells leads to a disease switch from AML to B-ALL, effectively changing the difficult-to-treat drug-resistant AML to another disease with more favorable CR (B-ALL). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IDH1 and IDH2 mutations in patients with acute myeloid leukemia: Suitable targets for minimal residual disease monitoring?

ObjectivesMolecular screening plays a major role in prognostic categorization and subsequent definition of treatment strategies for acute myeloid leukemia. The possibility of using IDH1/2 mutations as a marker for the monitoring of minimal residual disease (MRD) is still under investigation and remains unclear. MethodsIn this retrospective study, we evaluated 90 patients with de novo AML using Sanger sequencing (exon 4, IDH1 and IDH2). For subsequent MRD monitoring were used both methods, massive parallel sequencing and droplet digital PCR (ddPCR). ResultsWe identified 22 patients (24%) who harboured mutations in IDH1 or IDH2 genes. Fourteen (64%) of them had other commonly used MRD markers (insertion in NPM1 and partial tandem duplication of MLL, MLL-PTD). Eight of the 22 patients had IDH1 mutations, 13 had IDH2 mutations and 1 had both IDH1 and IDH2 mutations. In our cohort, this IDH1/2 marker responded to the treatment in all of the patients and reflected the onset of the relapse very well. NPM1 mutation based MRD monitoring was more sensitive and predicted relapse earlier but IDH1/2 based monitoring was more sensitive than a method based on MLL-PTD. Both massive parallel sequencing and ddPCR were competent to monitor MRD using IDH1/2. Nevertheless, ddPCR was able to achieve a higher sensitivity in some cases and moreover this method can analyse a single sample without significant price increases. ConclusionGiven these data, we conclude that IDH1/2 mutations can be used as a reliable and cost-effective marker for MRD monitoring.

Prognostic analysis of allogeneic hematopoietic stem-cell transplantation in 47 patients with acute myeloid leukemia and MLL rearrangement

目的研究混合谱系白血病（MLL）基因重排阳性急性髓系白血病（AML）患者行异基因造血干细胞移植（allo-HSCT）的预后特点。方法回顾性分析2009年9月至2016年5月于苏州大学附属第一医院行allo-HSCT的47例MLL基因重排阳性AML患者的临床资料。结果全部47例MLL重排阳性AML患者中男24例，女23例，中位年龄30（15~58）岁，M4/M5共36例（76.6%）。移植后2年总生存（OS）率为（64.4±8.4）%，无病生存（DFS）率为（47.3±9.3）%，复发率为41.0%，移植相关死亡率为17.9%。45例患者检出11q23易位，2例染色体核型正常患者检出MLL部分串联重复。t（6;11）组（16例）、t（9;11）组（15例）、其他类型组（16例）的2年OS率差异无统计学意义（χ2=1.509，P=0.472）。多因素分析显示，移植时年龄>45岁是影响OS的独立危险因素[HR=4.454（95%CI 1.314~15.099），P=0.016]，移植前MRD阳性是影响患者DFS[HR=4.236（95%CI 1.238~14.495），P=0.021]、复发[HR=5.491（95% CI 1.371~21.995），P=0.016]的独立不良预后因素，移植前疾病处于非CR状态患者移植相关死亡风险增高[HR=10.370（95%CI 1.043~103.110），P=0.046]。结论移植时年龄>45岁、移植前疾病处于非CR状态、移植前MRD阳性为影响allo-HSCT治疗MLL基因重排阳性AML患者预后的危险因素。

Abstract 2721: Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial

Abstract Introduction: Biomarker-driven early clinical trials of molecularly targeted agents have reported improvement of the response rate in various malignancies. In this pragmatic clinical trial, we aimed to assess whether biomarker-driven treatment strategy could improve outcomes of incurable hematologic cancer patients. Methods: This is an open-label, randomized, pragmatic trial in adult patients with hematologic cancer. Patients with any kind of hematologic cancer refractory to standard-of-care are eligible. We randomly assigned these patients to receive a matched molecularly targeted agent (experimental group) or treatment at physician’s choice (control group). Randomizations are determined by central block randomization in 2:1 ratio. Clinicians and patients are not masked to allocation. Whole exome and whole transcriptome sequencing (WES/WTS) are applied for detecting all classes of genomic alterations: single nucleotide variation (SNV), insertions and deletions (INDEL), copy number alterations, Fms-Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (ITD), Mixed-lineage leukemia partial tandem duplication and gene rearrangements. The primary endpoint is response rate in the intention-to-treat population. Results: Twenty-three patients with median age of 53 years and male to female ratio of 11:12 were enrolled between December 2015 and October 2016; 15 patients in experimental group, 8 patients in control group. Actionable mutations were identified in four patients in experimental arm. A 78-year-old female with acute myelogenous leukemia (AML) had FLT3 ITD at exon 14, which was identified as a long insertion mutation in SNV/INDEL calling. She received ASP2215 (FLT3 inhibitor), and showed a dramatic reduction in blast counts of peripheral blood. A 75-year-old female with myelodysplastic syndrome had TSC1P39A mutation. Because mTOR exists in downstream signaling pathway of TSC1, everolimus was administered and regression of disease activity, reflected by reduction of C-reactive protein level, was observed. NOTCH2 c.17′ and KITD815V mutation were identified in two patient; 69-year-old female with multiple myeloma and 42-year-old male with AML, respectively. Among 8 patients in control arm, seven patients received cytotoxic chemotherapy. One undergone another comprehensive genomic profiling called FoundationOne Heme, however no actionable mutations was identified. Conclusions: Among patients with incurable hematologic malignancy, actionable mutations could be identified in one fourth of patients for whom WES/WTS were performed. They showed favorable initial response to molecularly targeted agents. This randomized pragmatic clinical trial is currently ongoing(NCT02758080). Citation Format: Sanga Kim, Ryul Kim, Youngil Koh, Choong Hyun Sun, Hogune Im, Hong Seok Yun, Dong-Yeop Shin, Inho Kim, Sung-soo Yoon. Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2721. doi:10.1158/1538-7445.AM2017-2721

Abstract 2817: Enriched environment inhibits progression of acute myeloid leukemia

Abstract Background: Environmental factors and lifestyle have profound effects in the initiation, promotion and progression of cancer. Our recent work on environmental enrichment (EE), a housing environment boosting mental health, has revealed a novel phenotype characterized by a robust reduction in adiposity, resistance to diet-induced obesity (DIO), enhanced insulin sensitivity, lower serum IGF-1 and leptin levels, higher serum adiponectin level, enhanced immune functions, and inhibition in melanoma and colon cancer growth. We have teased out one key mechanism underlying the anticancer effect of EE: the stimulation of brain-derived neurotrophic factor (BDNF) expression in the hypothalamus and the subsequent activation of a specific neuroendocrine axis, the hypothalamic-sympathoneural-adipocyte (HSA) axis. However, the EE’s effects on hematologic malignancy are largely unknown. Here we generalized this intervention to acute myeloid leukemia (AML) using the MLL-partial tandem duplication (PTD) and Flt3-internal tandem duplication (ITD) double knock-in mouse (MllPTD/WT:Flt3ITD/WT) that recapitulates features of counterpart human AML. Methods: For transplantation of AML cells, Ly5.2 spleen cells isolated from leukemic MllPTD/WT: Flt3ITD/WT mice were resuspended in PBS. Syngenic Ly5.1 recipient C57Bl/6 male mice (CD45.1+) were sublethally irradiated (400 cGy). Five to six hours later, leukemic cells (4×106 per mouse) were injected through the lateral tail vein. Those mice were randomly assigned to either standard laboratory cage (SE) or EE housing. To monitor AML progression, white blood cell (WBC) counts were measured from peripheral blood. Survival experiments were performed. Results: EE mice had a median survival 14% longer than SE mice (SE = 36 ± 1.1 days, EE = 41 ± 1.2 days, p = 0.038) and improved hazard rate (log-rank test z = 1.99, p = 0.047). Based on WBC counts (&amp;gt;10.9 k/µL), 100% of SE mice and 80% of EE mice had apparent AML at three weeks post injection. At this time point, EE had a substantially lower WBC counts (SE = 76.9 k/µL, EE = 26.4 k/µL, p = 0.0006). WBCs from EE mice had lower neutrophil, monocytes, eosinophils and basophils counts, but had similar lymphocytes, resulting in a reduced neutrophil to lymphocyte ratio. Red blood cell (RBC) and platelet data were the same except for a slightly reduced mean corpuscular hemoglobin concentration (MCHC) in EE mice. At four weeks, EE mice were substantially more active and mobile than SE mice. At five weeks, at which point 3 SE and 5 EE mice remained alive, WBC counts remained lower in EE mice. Conclusions: This preliminary work demonstrated EE substantially improved progression-free survival and conferred a significant survival benefit. We will elucidate the underlying mechanisms of EE’s anti-AML effect by assessing metabolic modulation and immune regulation. Citation Format: Run Xiao, Stephen M. Bergin, Michael A. Caligiuri, Lei Cao. Enriched environment inhibits progression of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2817. doi:10.1158/1538-7445.AM2017-2817

A Novel Regimen for Acute Myeloid Leukemia with MLL Partial Tandem Duplication: Results of a Phase 1 Study NCI 8485

Background: MLL partial tandem duplication (MLL PTD) occurs in approximately 5-8% of patients with acute myeloid leukemia (AML) and is associated with an adverse prognosis. Our group has published that the MLL wild type (WT) allele is epigenetically silenced in MLL PTD; we showed that re-expression of this gene can be induced with methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors. Further, re-expression of MLL WT following combined decitabine and HDAC inhibitor treatment sensitized MLL PTD myeloid leukemia cells to chemotherapy in vitro. We hypothesized that epigenetic silencing of the MLL WT contributes to MLL PTD-associated leukemogenesis and that its pharmacologic re-expression with DNMT and HDAC inhibitors would activate apoptotic mechanisms important for chemo-response in the clinic. We aimed to develop a regimen to be tested in this unique molecular subset of disease. Because of the relatively low frequency of MLL PTD AML, this dose finding study was conducted in relapsed/refractory (R/R) AML regardless of molecular subtype but was enriched for MLLPTD.Methods:In this phase 1 study, adults aged 18-59 years with R/R AML, ECOG 0-2, and preserved organ function were enrolled. Patients received decitabine 20mg/m2 daily on days 1-10 and vorinostat 400mg daily on days 5-10 for all dose levels. Dose-escalated cytarabine was given on days 12, 14, 16 (six doses) according to the following schedule: dose level (DL) 1, 1.5g/m2/q12hr; DL 2, 2g/m2/q12hr; DL 3, 2.5g/m2/q12hr; and DL 4, 3g/m2/q12hr. Standard method 3+3 phase I design was used with the primary objective to determine the maximum tolerated dose and define specific toxicities with this combination therapy, in order to ultimately develop a regimen for MLLPTD AML.Results:Seventeen adults with R/R AML and median age of 46 years (range, 21-59 years) enrolled. The median number of prior induction therapies was 2 (range, 1-4). European LeukemiaNet (ELN) genetic risk classification frequencies were: favorable (n=2), intermediate-I (n=3), intermediate-II (n=5), and adverse (n=7), respectively. Four patients had MLL PTD. A total of 6 patients were treated at DL 1 with no non-hematologic dose limiting toxicity (DLT). DL 1 was expanded after two patients experienced prolonged but uncomplicated myelosuppression. Since both patients achieved complete remission (CR) shortly after passing the day 42 DLT cut off for hematologic recovery, the protocol was amended to allow further time for count recovery (up to 56 days). Three patients each were treated on DL 2 and 3; 5 patients were treated on DL 4 with no other DLTs observed. Diarrhea, nausea, fatigue, febrile neutropenia, and elevated ALT were the most common toxicities (any grade, regardless of attribution), occurring in 41%, 29%, 29%, 35%, and 35% of patients, respectively, but none were DLTs. In regards to ≥ Grade 3 toxicities, febrile neutropenia and catheter-related infections were most common at 35% and 24%. Significant mucositis was not observed. DL 4 was the recommended phase 2 dose (RP2D). CR or CR with incomplete count recovery (CRi) was observed in 6/17 patients (35%, 5 with CR). Of 6 responders, all (n=4) patients with abnormal karyotype achieved cytogenetic remission. The median number of prior therapies for patients achieving CR/CRi was 2 (range, 1-3). Four patients subsequently received allogeneic transplantation. Of the four patients known to have MLL PTD mutations, two responded (1 with CR and 1 with CRi). It is interesting to note that the two patients with MLL PTD mutations who did not respond to treatment also had FLT3-ITD mutations, while this mutation was absent in the two responding patients who had MLLPTD.Conclusions: We successfully determined the RP2D for this novel treatment regimen. The regimen had modest toxicities beyond uncomplicated (though prolonged) myelosuppression, and we propose that the study provides a framework for larger efficacy studies for AML patients with the uncommon but biologically distinct molecular feature of MLLPTD.Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA076576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Also supported by P30 CA016058/CA/NCI. DisclosuresNo relevant conflicts of interest to declare.

The Role of STAT5 in HOXA9-Associated Leukemia

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis.Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo.To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of MLL Partial Tandem Duplication in Acute Myeloid Leukemia of Intermediate Cytogenetic Risk: A Subgroup Analysis of Cetlam Protocol 2003 & 2012

BackgroundCytogenetics at diagnosis is the most important prognostic factor in acute myeloid leukemia (AML). Of note, intermediate cytogenetic risk group (IR-AML) is a very heterogeneous subset including normal karyotypes and all the cytogenetic abnormalities not included in the favorable or the adverse groups. Molecular alterations affecting NPM1, FLT3 and CEBPA show a prognostic impact in IR-AML. MLL partial tandem duplications (MLL-PTD) have also been described in this group of AML, but their prognostic impact have not been well established.AimTo analyze the prognostic relevance of MLL-PTD in the subset of patients diagnosed with IR-AML since 2003, and included in the CETLAM protocols LMA-2003 and LMA-2012.MethodsBetween 2003 and 2004 MLL-PTD were analyzed by Southern Blot (enzymes employed BglII, EcoRI, BamHI). Since 2004, a long PCR strategy was used to identify this abnormality.ResultsNPM1 mutations (NPM1mut), FLT3 internal tandem duplications (FLT3-ITD) and MLL-PTD were available for 893 patients. No MLL-PTD was found among 111 and 161 patients of the good and poor cytogenetic risk groups, respectively. The IR-AML group included 621 patients, and 37 carried a MLL-PTD (6%), thus only this cytogenetic group of patients was analyzed. NPM1mut were found in a 41% of patients and none of them had a concomitant MLL-PTD (p<0.001). FLT3-ITD were found in a 31% of patients, and 14 patients had also an MLL-PTD. No correlation between MLL-PTD and age, leukocyte count, and percentage of blasts in bone marrow was found. There was a significant association with gender: men were more frequently mutated than women (29 vs 8; p=0.001). Regarding outcome of IR-AML, leukemia-free survival (LFS) was significantly higher for patients without MLL-PTD (5-year LFS 44±3% vs 18±8%; p<0.001), and overall survival (OS) was also better for this subgroup of patients (5-year OS 42±2% vs 20±7%; p=0.004). There were no differences in the complete response rate, but patients with MLL-PTD had a higher risk of relapse (cumulative incidence at 5 years 39 vs 74%, p=0.000151). Among patients with MLL-PTD, no differences were observed depending on the concurrence of FLT3-ITD. When only patients with NPM1 wild-type were considered, MLL-PTD maintained a significantly poor prognostic impact (36±3% vs 21±7%; p=0.009).ConclusionsMLL-PTD is a genetic alteration found in a 6% of IR-AML. Patients with this abnormality have a worse LFS and OS than the rest of patients of the IR-AML group.Based on these results, patients with MLL-PTD should be considered as patients with poor cytogenetic risk AML for treatment allocation. DisclosuresNo relevant conflicts of interest to declare.

Abnormalities Detected By Array CGH and Fluorescence in Situ Hybridization in AML with Normal Karyotype Lacking Mutations in NPM1, CEBPA, RUNX1 and MLL Partial Tandem Duplications Are Associated with Unfavorable Outcome

Background: AML is a group of genetically distinct entities which have been defined by the presence of certain recurrent, mutually exclusive genetic abnormalities such as AML-specific fusion genes (e.g. PML-RARA, RUNX1-RUNX1T1) or recurrent molecular mutations (NPM1 mutations and CEBPA double mutations (dm)). In addition partial tandem duplications within the MLL gene (MLL-PTD) and RUNX1 mutations seem to play an important role in certain AML subtypes. A subset of AML with normal karyotype lacking the above-mentioned mutations is still poorly characterized. Array CGH and fluorescence in situ hybridization are able to detect abnormalities which are undetectable by chromosome banding analysis either due to a higher resolution, ability to detect copy neutral loss of heterozygosity (CN-LOH) and independence of in vitro proliferation.Aims: 1. Search for submicroscopic copy number changes and cryptic rearrangements in AML with normal karyotype lacking NPM1 and RUNX1 mutations, CEBPA dm, and MLL-PTD. 2. Determine whether submicroscopic cytogenetic changes impact on survival.Patients and Methods: For 1473 AML cases with normal karyotype complete data on mutation status of NPM1, CEBPA, RUNX1 and MLL -PTD was available. Of these 303 cases (21%) did not carry one of these mutations. Out of these 159 cases with de novo AML (median age: 68 years (range: 19-93)) were selected on the basis of availability of material for array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and FISH screening with probes for MLL, RUNX1, CBFB, NUP98, MECOM/EVI1, NPM1, ETV6 and DEK-NUP214 (MetaSystems, Altlussheim, Germany; ABBOTT, Wiesbaden Germany).Results: In total in 67 of 159 patients (42%) abnormalities were identified by FISH and/or array CGH. In detail, 12 balanced rearrangements were detected by FISH screening involving NUP98 (n=7, in 6 of these a NUP98-NSD1 rearrangement was identified by PCR), MLL (n=2) and MECOM, RUNX1 and CBFB (one each). In addition, 27 gains, 42 losses and 41 copy neutral losses of heterozygosity (CN-LOH) were observed in 58 (37%) patients. Recurrent gains affected regions 6q23.3q23.3 (135.325.751-135.607.060) (n=2) and 8q24.21q24.21 (130.517.732-130.808.381) (n=2) while recurrent losses were found for the regions 21q22.12q22.12 encompassing the RUNX1 gene (36.228.735-36.303.952) (n=5), 5q31.2q31.2 including i.a. EGR1 and CTNNA1 (137.617.569-138.993.959) (n=3), 2q34q34 including i.a. IKZF2 (213.371.237-214.560.488) (n=2), 7q22.1q22.1 encompassing i.a. CUX1 (100.485.221-101.916.623) (n=2), and Yq11.223q12 (24.980.949-28.804.541) (n=2). Recurrent CN-LOH were observed on chromosomes 11q (n=10), 2p (n=5), 4q (n=4), 21q (n=3), 1p (n=2), 17q (n=2) and 18q (n=2). 20/27 gains and 38/42 losses were < 10 megabases in sizes and thus below the resolution of chromosome banding analysis. Only in 7 (4%) patients abnormalities (n=11) were identified which in principle are detectable by chromosome banding analysis. These were missed by chromosome banding analysis due to insufficient in vitro proliferation of the aberrant clone, small clone size or poor chromosome morphology. Comparing age, white blood cell count and bone marrow blast counts revealed no differences between patients with or without abnormalities detected by FISH and/or array CGH. However, FLT3-ITD and WT1 mutations were more frequent in cases with abnormalities (25% vs 7%, p=0.001; 9% vs 1%, p=0.021). Survival analysis was performed for 90 intensively treated patients. Overall survival (OS), event-free survival (EFS) and OS with censoring at time of allogeneic SCT (OSctx) were significantly shorter in patients with abnormalities detected by FISH and/or array CGH compared to those without (median OS: 19 vs 49 months, p=0.027; Figure 1; median EFS: 9 vs 21 months, p=0.016; median OSctx: 13 vs 26 months, p=0.018).Conclusions: 1. AML with normal karyotype based on chromosome banding analysis lacking a disease defining molecular mutation harbor balanced rearrangements, copy number gains and losses as well as CN-LOH at a high frequency (42%). 2. The presence of these abnormalities has a negative impact on survival demonstrating that FISH and array CGH can add prognostic information in the diagnostic work-up of AML with normal karyotype lacking a disease defining mutation. 3. Further investigations of mutations in genes located in regions of recurrent CN-LOH is necessary. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zieschang:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

HIF-1a Pathway, As a Signal Funnel for Genetic, Epigenetic, and Metabolic Aberrations, Is Sufficient and Essential for MDS Development

Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematopoietic disorders, which are characterized by ineffective hematopoiesis and uni- or multi-lineage dysplasia. Despite the fact that a variety of genetic, epigenetic, and metabolic aberrations have been identified, the clinical features for MDS remain similar, indicating that there is a common underlying mechanism for MDS pathogenesis. Accumulating clinical and research evidence has shown the involvement of systemic inflammation and activated immune signaling in MDS pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is a critical transcription factor for the hypoxic response, angiogenesis, and cancer development. Importantly, HIF-1α is also a key regulator for immune cell activation.To determine whether MDS patients have an activated HIF-1α signature, we analyzed a public gene expression array data set of CD34+ BM cells from a large cohort of MDS patients (n = 183). Although HIF-1α mRNA remains unchanged, some HIF-1α target genes relating with survival/cell growth, glucose metabolism, and invasion/metastasis were significantly activated in the broad spectrum of MDS. We further determined HIF-1α expression in the BM biopsies from MDS patients (n = 39) using immunohistochemistry staining. We found a high frequency of HIF-1α expressed cells in both low- and high-risk IPSS groups. Consistent with this human data, deficiency of frequently mutated genes in MDS resulted in an activated HIF-1α signature in mice. We found that the levels of Hif-1α protein in the c-Kit+ BM cells from Dnmt3a-KO, Runx1-KO, and Mll-partial tandem duplication (PTD) knock-in (MllPTD/WT) mice were significantly upregulated compared with those from WT controls. Although the expression level of Hif-1α protein in the c-Kit+ BM cells from Tet2-KO mice was not changed, they had an activated HIF-1α signature. IDH1/2 mutations are also known to elevate Hif-1α proteins. These results suggest that the HIF-1α pathway is widely activated in MDS patients by MDS-genic mutations through different mechanisms.To determine whether HIF-1α is sufficient for developing MDS phenotypes, we generated blood specific inducible HIF-1α transgenic mice. Using Vav1-Cre/Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-1α can be induced in a doxycycline dependent manner. HIF-1α-induced mice developed thrombocytopenia, leukocytopenia, and macrocytic anemia. We found micromegakaryocyte and multi-segmented megakaryocyte formations which are a major diagnostic criteria for MDS. In the BM of anemic mice, we found significant reduction of basophilic and polychromatic erythroblasts. The central macrophages are known to form erythroblastic islands that play a critical role in the late stage of erythropoiesis. Both the frequency and absolute number of F4/80+ Ter119+ BM erythroblastic islands were significantly decreased in HIF-1α-induced mice compared to the control mice. We also found activation of both innate and adaptive immunity in HIF-1α-induced mice. Lineage specific HIF-1α induction revealed the cell-intrinsic effect of HIF-1α on aberrant megakaryocytopoiesis and cell-extrinsic effect of HIF-1α on macrocytic anemia development.We have previously shown that MllPTD/WT mice develop several MDS-associated features, such as increased self-renewal and apoptosis in HSPCs, expansion of myeloid progenitors, and skewed myeloid differentiation. Synergistic effects between MLL-PTD and RUNX1 mutant (S291fs) or Runx1 deletion, further accumulated the Hif-1α protein and caused a variety of MDS features that phenocopy the human MDS. To understand the role of HIF-1α in the context of MDS pathogenesis, we took genetic and pharmacologic approaches. Hif-1α deletion significantly abrogated disease development and rescued macrocytic anemia and thrombocytopenia. This is not due to the elimination of donor derived cells. After the deletion of Hif-1α, a majority of donor derived cells in PB were still CD11b+ cells. HIF-1α inhibition significantly reduced colony formation of MllPTD/WT/RUNX1-291fs cells and MllPTD/WT/Runx1D/D cells in vitro, and prolonged survival of MDS mice in vivo.In conclusion, we identified the sufficient and essential role of HIF-1α in developing MDS. These findings implicate that HIF-1α pathway may be the common underlying mechanism of MDS pathophysiology and could be an effective therapeutic target for a broad spectrum of MDS patients. DisclosuresShih:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Phase 1 Study of the DOT1L Inhibitor, Pinometostat (EPZ-5676), in Adults with Relapsed or Refractory Leukemia: Safety, Clinical Activity, Exposure and Target Inhibition

Introduction: Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) result in recruitment of another HMT, DOT1L, to a multi-protein complex. This leads to abnormal methylation of Histone H3 lysine 79 (H3K79) at MLL target genes and enhanced expression of leukemogenic genes such as HOXA9 and MEIS1 ( Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Pinometostat treatment of MLL-rearranged cells and xenografts reduced histone H3K79 methylation, decreased MLL target gene expression, and induced selective leukemia cell kill (Daigle, 2013). We report the safety, activity, pharmacokinetics (PK) and pharmacodynamics (PD) in the phase 1 trial of pinometostat in adult patients (pts) with relapsed/refractory (R/R) leukemia.Methods: This open label dose escalation/expansion study of pinometostat enrolled pts >18 years (yrs) with R/R leukemia (NCT01684150). In the dose-escalation phase, pts with AML, ALL, mixed lineage leukemia (MLL), myelodysplastic syndrome, myeloproliferative neoplasm or chronic myeloid leukemia were eligible. Eligibility in the two expansion cohorts: 90 mg/m2 (n = 17) and 54 mg/m2 (n = 6), was restricted to pts with MLL-r or MLL-partial tandem duplication (MLL-PTD). Pinometostat was given via continuous intravenous infusion (CIV) for 21 of 28 day cycles in the dose escalation phase and CIV for 28 of 28 day cycles in the expansion phases, until disease progression or unacceptable toxicity. All pts underwent serial collection of PK and peripheral blood mononuclear cells (PBMC) for PD. Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for dimethylation of H3K79 (H3K79-me2) by ChIP-Seq.Results: As of 28-June-2015, 49 pts have enrolled in the dose escalation and expansion phases. Pts receiving 21/28 day infusions: 12 (n=1), 24 (n=5), 36 (n=4), 54 (n=6) and 80 mg/m2/day (n=3). Pts receiving 28/28 day infusions: 54 (n=6) and 90 mg/m2/day (n=24).Table 1Patient Characteristicsn (%)Median age, yrs (range)51 (19 - 81)Sex (M / F)27/22DiagnosisMLL-r*29 (59)AMLMLL-PTD**5 (9)MLL-wt7 (14)ALLMLL-r*5 (10)MLL-wt1 (2)MLLMLL-r1 (2)CMMLMLL-r1 (2)# of prior therapeutic regimens1 - 229 (59)3 - 418 (36)>42 (4)Prior allogeneic hematopoietic cell transplant20 (41)* centrally confirmed by karyotype/FISH** centrally confirmed by NGS (next generation sequencing)Adverse events (AEs) reported in >15% of pts regardless of attribution were: nausea, constipation, vomiting, abdominal pain, diarrhea, hypocalcemia, hypokalemia, hypomagnesemia, fatigue, fever, peripheral edema, mucositis, febrile neutropenia, leukocytosis, anemia, cough, dyspnea, and pneumonia. Grade ≥3 non-hematologic related toxicities include: hypophosphatemia (n=1), decreased ejection fraction (n=3), or elevated transaminases (n=1). Nine patients had leukocytosis (absolute monocyte and neutrophil 50% above baseline and above upper limit of normal) or differentiation. The median days of pinometostat treatment was 35 days (range 3-189 days). To date, objective responses observed are morphologic CR (1 pt), cytogenetic CR (MLL negative by FISH) (1 pt), PR (1 pt) and resolution of leukemia cutis (3 pts). Dose proportional PK was observed with rapid attainment of steady-state plasma concentrations (Css) on Day 1 of treatment. Plasma Css correlated with inhibition of global H3K79-me2 in PBMCs. H3K79-me2 ChIP-Seq demonstrated pinometostat induced reductions in methylation at MLL -r target genes HOXA9 and MEIS1 (median inhibition = 61%: range = 13-91%) in all 9 pts analyzed to date from the 90 mg/m2 expansion cohort. Inhibition of H3K79-me2 in leukemic blasts is consistent with DOT1L suppression. PK-PD relationships in both expansion cohorts using both free and total plasma Css are being explored.Conclusions: Pinometostat administered as a CIV to adults with R/R leukemia has an acceptable safety profile. Clinical activity as demonstrated by both marrow responses and resolution of leukemia cutis were observed. In addition, analysis of H3K79-me2 by ChIP-Seq demonstrated PD reductions in the methylation of MLL-r target genes following pinometostat exposure, as expected from DOT1L inhibition. Relationships between PK exposure, reductions in pinometostat induced H3K79-me2 levels and clinical response are being interrogated. DisclosuresStein:Seattle Genetics: Consultancy; Agios Pharmaceuticals: Consultancy. Rizzieri:Teva: Other: ad board, Speakers Bureau; Celgene: Other: ad board, Speakers Bureau. Berdeja:Novartis: Research Funding; Takeda: Research Funding; BMS: Research Funding; Curis: Research Funding; MEI: Research Funding; Abbvie: Research Funding; Onyx: Research Funding; Array: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Acetylon: Research Funding. Altman:Novartis: Other: Advisory board; Spectrum: Other: Advisory board; Ariad: Other: Advisory board; Seattle Genetics: Other: Advisory board; BMS: Other: Advisory board; Astellas: Other: Participation in an advisory board December 2013. Thomson:Epizyme, Inc: Employment. Blakemore:Epizyme: Employment. Daigle:Epizyme, Inc: Employment. Fine:Epizyme: Employment. Waters:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Ho:Epizyme, Inc: Employment.

High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

Abstract B06: Distinct genomic profiles and targetable alterations revealed by FoundationOne® Heme in hematolymphoid malignancies in adolescents and young adults.

Abstract Background: Hematolymphoid malignancies (HLM) in adolescent and young adult (AYA, age 15-39) patients exhibit distinct clinical and routine pathologic characteristics. Comparative analysis of genomic alterations (GA) of HLM in AYA versus younger and older patient cohorts has not been reported previously. Methods: DNA was extracted and captured using custom baits targeting all exons of 405 cancer-related genes and select introns of 31 genes commonly rearranged in cancer (n=24), or both DNA and RNA were extracted and captured using custom baits for 405 genes and 265 frequently rearranged genes, respectively (n=49) (FoundationOne® Heme). All captured libraries were sequenced to high uniform depth in a CLIA-certified, CAP-accredited laboratory with an average depth of &amp;gt;500x for DNA and &amp;gt;10M unique pairs for RNA (Illumina HiSeq). Sequence data were assessed for base substitutions, insertions/deletions (indels), copy number alterations, and select gene rearrangements. Results: The assays identified an average of 3.1 cancer-related GAs per HLM (range 0-22), including 135 base substitutions, 45 indels, 36 gene fusions or rearrangements, 13 gene deletions, and 12 gene amplifications. The most frequently altered genes in this AYA HLM cohort were TP53 (22%), NRAS (12%), KRAS (11%), PTPN11 (10%), MYC (8%), B2M (8%), SOCS1 (7%), FLT3 (7%), and CDKN2A (7%). Clinically relevant GAs (targetable by therapies approved or in clinical trials) were found in 64% of cases, including 60% of lymphoma, 71% of acute myeloid leukemia (AML), and 79% of acute lymphoblastic leukemia (ALL). Clinically relevant GAs identified in this AYA cohort included NRAS and KRAS (23%), targetable by MEK inhibitors, FLT3 (7%), targetable by multiple agents, as well as partial tandem duplication of MLL and rearrangement of IGH/MYC. AML AYA samples contained a high frequency of PTPN11 mutations (41%) which can activate the RAS/MAP/ERK, PI3K, and Src pathways. The enrichment of PTPN11 mutations in 7/17 (41%) AYA AML vs. 0/10 (0%) pediatric AML (age&amp;lt;15) and 1/ 80 (1%) of older adult AML was significant (p=0.0001). A higher frequency of KRAS mutations in AYA AML (29%) vs. older adults (6%) was additionally observed (p=0.01). 2 of 9 AYA diffuse large B-cell lymphoma (DLBCL) cases were found to harbor ALK fusions, a potential enrichment relative to older patients which will be further explored. Conclusions: FoundationOne Heme can detect all classes of genomic alterations in AYA hematolymphoid malignancies. Significant molecular distinctions identified in this AYA population implicate age-related mechanisms in the development and progression of HLM, and provide novel potential therapeutic approaches to improve clinical outcomes in this distinct patient cohort. Citation Format: Kai Wang, Deborah Morosini, Roman Yelensky, Doron Lipson, Juliann Chmielecki, Siraj M. Ali, Jeffrey S. Ross, Phil Stephens, Vincent A. Miller. Distinct genomic profiles and targetable alterations revealed by FoundationOne® Heme in hematolymphoid malignancies in adolescents and young adults. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B06.

The transcriptomic landscape and directed chemical interrogation of MLL-rearranged acute myeloid leukemias.

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.