ALBERT

Description: Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients. To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (NSG) mice. All samples engrafted and expanded in NSG mice, leading to overt leukemia with a latency of 49 to 276 days. Purification of leukemic blasts from a single moribund mouse yielded on average 108 cells, providing sufficient material to screen large numbers of compounds. In vitro conditions were defined that support growth in 40% of the patient specimens, allowing for a more accurate determination of drug sensitivity. Growth in vitro was associated with early onset of disease in NSG xenografts and younger age at presentation, allowing us to evaluate patient samples that represent aggressive high risk disease. Using this system, we tested bortezomib in addition to 28 other drugs, including standard ALL therapeutic agents as well as targeted kinase inhibitors and inhibitors of epigenetic marks. Three classes of agents were active in this system: anthracyclines, histone deacetylase inhibitors (HDACi), and the proteasome inhibitor bortezomib. In contrast to anthracyclines and HDACi, where IC50 values were on par with those reported in the literature for primary childhood ALL samples, MLLr infant samples required 10-100 fold less bortezomib to induce toxicity. Bortezomib has been shown to mediate responses through several mechanisms, including NFKB inhibition, stabilization of cell cycle regulatory proteins, and induction of apoptosis. Recently, proteasome inhibition has been demonstrated to lead to accumulated MLL fusion protein levels, triggering apoptosis and cell cycle arrest in MLLr cell lines. To determine if NFKB inhibition also plays a role, we evaluated cellular concentrations of the activated NFKB transcription factor, but failed to see decreased levels when MLLr cells were treated with bortezomib. Bortezomib has also been shown to deregulate ubiquitin stores and deplete histone H2B ubiquitination (H2Bub), an epigenetic mark that is linked to histone methylation and expression. Recently, several groups have demonstrated that H2Bub is required for DOT1L activity and HOX gene expression. We therefore evaluated H2Bub levels in bortezomib-treated patient samples and confirmed depletion of this epigenetic mark. Furthermore, patient samples treated with bortezomib downregulated both the MLL gene expression signature and signatures of downstream targets, such as cMYC, demonstrating that the MLL transcriptional program is inhibited in the presence of bortezomib. ChIP-seq is underway to map H2Bub and H3K79 methylation changes genome wide in response to treatment with bortezomib. The HDACi vorinostat and bortezomib have both been evaluated in Phase I and II pediatric leukemia clinical trials. Based on the safety and efficacy from these earlier studies, we treated 6 relapsed/refractory MLLr leukemia patients with a chemotherapy regimen that included mitoxantrone, vorinostat, and bortezomib. 4 patients had a complete response (CR), 1 patient had a partial response (PR) and 1 patient had stable disease for an overall response rate of 5/6 (83%). Clinical trials are in development to assess this combination further for both relapsed MLLr disease as well as newly diagnosed infant ALL. Our data suggests that these three classes of drugs, identified in our laboratory assays, are clinically active thus validating our system. We are now using this platform to proceed with a high throughput drug screen to identify additional compounds for future clinical development. Disclosures Off Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.

Description: Key Points Using a mouse genetic mouse model of Ph+ B-lineage ALL, endogenous antiapoptotic MCL-1 is required for leukemia survival. In BCR-ABL+ B-lineage ALL human and mouse cells, combining TKIs with small-molecule inhibitors of BCL-2 can potentiate sensitivity to cell death.

Description: Acute megakaryoblastic leukemia (AMKL) accounts for ~10% of childhood AML. AMKL patients without Down syndrome have a poor outcome with a 3 year survival of less than 40%. To gain insight into the biology of this disease, we previously performed transcriptome sequencing on diagnostic blasts from a discovery cohort of 14 pediatric cases and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult samples. This analysis identified novel fusion transcripts restricted to pediatric AMKL including CBFA2T3-GLIS2,GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9. To confirm their role in oncogenesis and gain insight into the mechanism whereby these fusions promote disease, we introduced each of them into murine hematopoietic cells and assessed their effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a control retrovirus failed to form colonies after the second replating. By contrast, expression of each of the fusion genes resulted in a marked increase in self-renewal capacity, with colony formation persisting through 10 replatings. Immunophenotypic analysis revealed evidence of megakaryocytic differentiation in CBFA2T3-GLIS2 and MN1-FLI1 cohorts, whereas NIPBL-HOXB9 and GATA2-HOXA9 cells carried markers consistent with myeloid progenitors. Transplantation of fusion gene modified bone marrow cells into syngeneic recipients induced overt leukemia in all cohorts with the exception of CBFA2T3-GLIS2, suggesting an essential requirement for cooperative mutation(s) in cases expressing this chimeric gene. To assess self-renewal activity of the leukemia generated in our murine models, we conducted secondary transplants for all cohorts. In all cases, the leukemia was transplantable with a shorter latency than in the primary transplant setting. To characterize the tumors at the molecular level, 5 samples from each of the 3 fusions underwent array comparative genomic hybridization, transcriptome, and whole exome sequencing. Samples demonstrated a small number of cooperating mutations with 1.5 copy number alterations (range 0-6) and 6.4 single nucleotide variations (range 2-13) per case. Overall, cases carried an average of 7.9 mutations (range 2-14). Despite the low number of lesions, recurrently mutated genes were identified. These include activating mutations in Flt3, Kras, and cMet, as well as loss of function mutations in the tumor suppressors Phactr4, Wt1, and Tet2. A comparison between fusion subtypes did not reveal any statistically significant differences, although there was a trend towards a greater number of mutations in the GATA2-HOXA9 cohort. Transcriptome sequencing of cohorts, along with normal hematopoietic progenitor subsets, confirmed unique gene expression patterns between each of the fusions. Consistent with immunophenotyping, MN1-FLI1 demonstrated enrichment of the MEP signature while NIPBL-HOXB9 and GATA2-HOXA9 were enriched for CMP and monocyte precursor signatures respectively. ChIP-seq analysis of each of the fusions is underway to definitively identify the genomic targets whose expression is directly altered by their binding. A common characteristic between all fusions is the presence of protein interaction domains contributed by the N term partner, and DNA binding domains contributed by the C term partner. To determine if these fusions have a novel gain of function distinct from their independent counterparts, we introduced each partner gene into murine bone marrow cells for transplantation experiments. As previously described, introduction of MN1 into hematopoietic cells led to a highly penetrant leukemia. In contrast, HOXA9, HOXB9, and FLI1 all had 〉75% disease free survival with few myeloid leukemias resulting from their over expression, while GATA2 failed to induce any disease at all. NIPBL’s size precluded transplant assays. Therefore, to evaluate its contribution we introduced a point mutation previously shown to disrupt binding of NIPBL to the cohesion component MAU2. This alteration abrogated the ability of the fusion to induce leukemia in our transplant model, demonstrating the importance of this interaction in the pathogenesis of disease. In conclusion, our data confirms a pathogenic role for GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9 in AMKL. Further studies delineating the cooperating mutations required for CBFA2T3-GLIS2 are indicated. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 757 Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%). To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. *TAG and ALG contributed equally to this work. Disclosures: Biondi: BMS, Novartis, Micromed: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding. Doehner:Hoffmann La Roche: Honoraria.