MLL-rearranged B-ALL Research Articles

The RNA Binding Protein IGF2BP3 Is Required for MLL-AF4 Mediated Leukemogenesis

Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

Correction: NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.

PO-139 Investigating unusual synthetic lethalgenes in acute lymphoblastic leukaemia

IntroductionB-cell acute lymphoblastic leukaemia (B-ALL) is caused by malignant transformation of B-cell progenitors and has multiple genetic subtypes, each characterised by specific genetic abnormalities, including MLL-rearranged and TCF3/PBX1 t(1;19). Survival for B-ALL is favourable compared to many other cancers, however, there is a need for novel therapeutic approaches that are less toxic and lead to better long term outcome for patients compared to current treatment options. These could potentially be found by identifying subtype-specific synthetic lethal (SL) genes. We have developed a novel bioinformatic approach which uses genome-wide methylation and expression data to identify candidate SL genes in specific genetic subtypes of disease. We aimed toidentify candidate SL genes in MLL-rearranged B-ALL, utilising our novel bioinformatics approach.Material and methodsGenome-wide methylation and expression data from patient samples were used to identify candidate genes. Candidate SL genes were knocked down with siRNA in cell lines with/without the corresponding subtype defining genetic abnormality. Apoptosis was measured by Annexin-V staining, and proliferation by cell counts using a specialised flow cytometer. Gene re-expression was performed using a lentiviral construct, co-expressing eGFP.Results and discussionsSurprisingly, in addition to a single candidate SL gene specific for MLL-fusion related B-ALL, our novel bioinformatic analysis also identified two classes of gene not generally seen in our analysis of other subtypes; a candidate subtype-specific tumour suppressor (KLF11) and a candidate shared SL gene (LAMP5, shared with t(1;19). LAMP5 knockdown significantly increased apoptosis (12.7%, p=0.0012) and decreased proliferation (23.3%, p=0.0006) in a t(1;19) cell line but not in MLL cell lines. As predicted, KLF11 re-expression in the MLL-rearranged SEM cell line caused significantly reduced cell growth/survival compared with the KLF11-negative parental cell line. However, high level re-expression in three cell lines expressing endogenous KLF11 resulted in similar effects on cell growth, implying that high level KLF11 expression was universally toxic in leukaemia cell lines.ConclusionLAMP5 may be a SL gene, however only specifically in t(1;19) B-ALL. This is the first time this gene has been linked to leukaemia and if confirmed to have a role in this subtype of B-ALL it would represent a novel therapeutic target.

Abstract 4958: VTP50469 is a novel, orally available menin-MLL1 inhibitor effective against MLL-rearranged and NPM1-mutant leukemia

Abstract Inhibition of the menin(MEN) and MLL1(KMT2A) interaction reverses leukemic gene expression driven by MLL-fusion proteins and is a potential targeted therapeutic strategy in MLL-rearranged AML. Recent data show that inhibition of this interaction could also be efficacious in NPM1-mutant AML. We developed potent and selective menin-MLL1 inhibitors that are highly selective and effective in vitro and in vivo. Structure-based design yielded a potent small-molecule inhibitor of the menin-MLL1 binding interaction, VTP-49477 (Ki=12+5 pM). Human and mouse leukemia cell lines driven by MLL-fusion proteins or NPM1 mutations were selectively sensitive to VTP-49477 (IC50 of ~10 nM). Optimization of the inhibitor structure led to an orally available inhibitor, VTP-50469 (Ki=104+30 pM). Similar to VTP-49477, human cell lines and mouse primary cells carrying MLL-fusions or NPM1c-mutations were highly sensitive to treatment with VTP-50469 (IC50 ~20 nM). Mechanistically, treatment of NPM1-mutant OCI-AML3 cells and MLL-AF9 rearranged MOLM13 cells with VTP-50469 caused >20-fold reduction of menin in high-molecular-weight protein complexes as assessed by glycerol gradient sedimentation. Concordantly, VTP-50469 treatment resulted in >10-fold reduction in menin chromatin occupancy as assessed by ChIP-seq in MLL-AF4 rearranged RS4;11 and MOLM13 cells. Furthermore, treatment with either VTP50469 or VTP4777 reduced expression of common MLL-fusion target genes in RS4;11 and MOLM13 cells and also modulated gene expression in NPM1-mutant leukemia cells. Human PDX models were treated with either VTP-49477 or VTP-50469. Treatment of MLL-rearranged B-ALL and AML PDXs (n=3) with VTP-49477 for 28 days (50mg/kg, bid, IP) resulted in significant reduction of leukemia burden (median: 2-fold in BM, 3-fold in SP, and 6-fold in PB). For oral dosing, VTP-50469 was formulated in mouse chow (0.1%, total daily dose ~175mg/kg/day) and PDX models were treated with this chow for 28 days. Mice engrafted with MLL-rearranged B-ALL, MLL-rearranged AML and NPM1-mutant AML PDXs (n=5) showed dramatic reductions of human leukemia cells in peripheral blood, spleen and bone marrow as compared to control treated mice (median: 225-fold in BM, 14-fold in SP and 129-fold in PB). AML cells showed increased differentiation with elevated expression of CD11b, CD13, CD86 and loss of common MLL-fusion target gene expression. Treatment of MLL1wt and NPM1wt B-ALL and AML PDXs with VTP-50469 did not reduce leukemia burden, confirming VTP-50469 menin-MLL1 target selectivity. Treatment with VTP-50469 did not alter normal peripheral blood counts and no weight loss was observed during treatment. Therefore, at a highly effective dose, VTP-50469 does not have detectable toxicity. Based on these data, inhibition of the menin-MLL1 interaction with VTP-50469 may have beneficial activity in up to 40% of human AML and most infant leukemias. Citation Format: Andrei V. Krivtsov, Benjamin K. Eschle, Matthew Witkin, Jayant Y. Gadrey, Hannah J. Uckelmann, Sayuri Kitajima, Gerard M. McGeehan, Scott A. Armstrong. VTP50469 is a novel, orally available menin-MLL1 inhibitor effective against MLL-rearranged and NPM1-mutant leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4958.

PO-142 RNAi-mediated therapeutic knockdown of the neuropilin axis and GIPC1 in pancreatic carcinoma

IntroductionB-cell acute lymphoblastic leukaemia (B-ALL) is caused by malignant transformation of B-cell progenitors and has multiple genetic subtypes, each characterised by specific genetic abnormalities, including MLL-rearranged and TCF3/PBX1 t(1;19). Survival for B-ALL is favourable compared to many other cancers, however, there is a need for novel therapeutic approaches that are less toxic and lead to better long term outcome for patients compared to current treatment options. These could potentially be found by identifying subtype-specific synthetic lethal (SL) genes. We have developed a novel bioinformatic approach which uses genome-wide methylation and expression data to identify candidate SL genes in specific genetic subtypes of disease. We aimed toidentify candidate SL genes in MLL-rearranged B-ALL, utilising our novel bioinformatics approach.Material and methodsGenome-wide methylation and expression data from patient samples were used to identify candidate genes. Candidate SL genes were knocked down with siRNA in cell lines with/without the corresponding subtype defining genetic abnormality. Apoptosis was measured by Annexin-V staining, and proliferation by cell counts using a specialised flow cytometer. Gene re-expression was performed using a lentiviral construct, co-expressing eGFP.Results and discussionsSurprisingly, in addition to a single candidate SL gene specific for MLL-fusion related B-ALL, our novel bioinformatic analysis also identified two classes of gene not generally seen in our analysis of other subtypes; a candidate subtype-specific tumour suppressor (KLF11) and a candidate shared SL gene (LAMP5, shared with t(1;19). LAMP5 knockdown significantly increased apoptosis (12.7%, p=0.0012) and decreased proliferation (23.3%, p=0.0006) in a t(1;19) cell line but not in MLL cell lines. As predicted, KLF11 re-expression in the MLL-rearranged SEM cell line caused significantly reduced cell growth/survival compared with the KLF11-negative parental cell line. However, high level re-expression in three cell lines expressing endogenous KLF11 resulted in similar effects on cell growth, implying that high level KLF11 expression was universally toxic in leukaemia cell lines.ConclusionLAMP5 may be a SL gene, however only specifically in t(1;19) B-ALL. This is the first time this gene has been linked to leukaemia and if confirmed to have a role in this subtype of B-ALL it would represent a novel therapeutic target.

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL

Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

SummaryInduced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

Anti-proliferative evaluation of monoterpene derivatives against leukemia

The cure rate of pediatric acute lymphoblastic leukemia (ALL) has significantly improved in the past thirty years, however not all patient cohorts respond well to current chemotherapy regimens. Among the high risk patient cohort is infants with MLL-rearranged (MLL-r) B-ALL, which remains dismal with an overall survival rate <35%. Our program is interested in identifying new molecular scaffolds to better understand the underlying mechanisms and ultimately provide new targeted treatments. Based on a phenotypic screen, phenolic natural products were identified as promising scaffolds for further chemical evaluation. Herein we disclose the effects of a potent anti-proliferative compound 31 against human ALL leukemia cellular models.

MLL-Rearranged Infant ALL, but Not Standard-Risk ALL, Induces Inflammasome Signaling and Immune Activation

T cell-based therapy for infants with MLL-rearranged B-ALL (MLL-ALL) represents a promising approach in this high-risk, poor-prognosis subset. Factors in the immune microenvironment that influence T-cell function, however, are poorly understood in MLL-ALL. We have observed complete inhibition of healthy donor T-cell proliferation by ALL cells from MLL-ALL, but not standard risk ALL (SR-ALL) patients. T-cell function is restored in the presence of bone marrow macrophages. In this study, we hypothesized that MLL-ALL cells selectively induce the inflammasome- a cytokine signaling complex formed by macrophages in response to damage-associated molecular patterns (DAMPs) released by leukemic cells. In the canonical inflammasome pathway, DAMPs engage macrophage Toll-like receptors, which activate caspase-1 and drive production of T-cell activating cytokines such as IL1-β.First, we determined whether ALL cells obtained from MLL-ALL vs. SR-ALL patients could produce high mobility group box 1 (HGMB1), a prototypical DAMP associated with tumor cell stress and macrophage PRR activation, particularly after anthracycline therapy. CD19+ cells were isolated from the diagnostic bone marrow specimens of patients with MLL-ALL (n=1), SR-ALL (n=4), and healthy donor PBMC (n=2) and treated with either control media or 2uM doxorubicin. Prior to treatment both MLL-ALL and SR-ALL, but not healthy donor cells, expressed HMGB1 protein as measured by Western immunoblot (29 kDa, GADPH internal control). Following doxorubicin treatment, HMGB1 protein was increased in MLL-ALL but not SR-ALL. Soluble HMGB1, measured by ELISA, was significantly increased in MLL-ALL (12.6 + 3.7 ng/ml untreated vs. 51.4 + 1.9 treated) compared to SR-ALL cells (5.0 + 0.8 ng/ml untreated, 4.3 + 1.5 treated, p<0.05). Soluble HMGB1 was not detectable in healthy donor cells before or after treatment.Next, we determined whether the increased HMGB1 secretion observed in MLL-ALL vs. SR-ALL cells was associated with macrophage caspase-1 activation. Control media and 2 uM doxorubicin treated-and-washed MLL-ALL, SR-ALL and healthy control CD19+ cells were cultured alone or 1:1 with allogeneic primary human macrophages for 24 hours. The active isoform of caspase-1 (20 kD tetramer) was detectable by immunoblot only in macrophages exposed to doxorubicin-treated MLL-ALL. Mean levels of IL1-β, measured by ELISA, were significantly increased in cocultures of doxorubicin-treated MLL-ALL + macrophages (1255 pg/ml + 322) compared to untreated MLL-ALL (820 pg/ml + 183, p=<0.05) and doxorubicin-treated SR-ALL + macrophages (494 pg/ml + 85 p<0.01). Macrophages cultured alone did not produce significant levels of IL1-β before or after doxorubicin treatment.Finally, we wished to determine whether expression of Toll-like receptor 4, a surface receptor for HMGB1 known to signal caspase-1, was increased on macrophages exposed to doxorubicin-treated MLL-ALL vs. SR-ALL. Folowing coculture with untreated and doxorubicin treated MLL-ALL and SR-ALL cells, macrophage TLR4 mRNA and surface expression was quantified by qPCR and flow cytometry, respectively. Both mRNA and surface expression of TLR4 was high at baseline, and did not change significantly after coculture. Unexpectedly, however, the MLL-ALL cells themselves displayed a significant increase in TLR4 mRNA (20-fold compared to endogenous beta-glucuronidase, p<0.0001; 12-fold compared to untreated MLL-ALL, p<0.05 by ΔΔCt) and surface expression (median fluorescence intensity 1005 + 89 vs. 367 + 32 respectively, p<0.05) following doxorubicin treatment. This phenomenon was not observed in SR-ALL or healthy CD19+ cells and is consistent with other tumor models of autocrine immune activation by HMGB1.Taken together, these data indicate that primary MLL-ALL cells, but not SR-ALL cells, are capable of inducing a caspase-1 associated inflammasome that enhances both innate and T-cell immune activation following anthracycline therapy. Studies are currently underway with additional primary MLL-ALL samples to determine how strategic augmentation of this pathway may impact the anti-leukemia killing capacity of therapeutic T-cells. DisclosuresNo relevant conflicts of interest to declare.

MLL-Rearranged B Lymphoblastic Leukemias Selectively Express the Immunoregulatory Carbohydrate-Binding Protein Galectin-1

Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL. Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation. Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells. In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.

Expression of Mir-196b Is Not Exclusively MLL-Driven but Especially Linked to Activation of HOXA Genes in Children with Acute Lymphoblastic Leukemia.

Abstract 580MicroRNAs (miRNAs) play important roles in diverse biological processes including hematopoiesis. As a consequence, aberrant expression of miRNAs may contribute to hematopoietic malignancies. It has been reported that miR-196b is transcriptionally activated by MLL and MLL-fusion genes and is therefore highly expressed in MLL-rearranged leukemia. In order to investigate whether high expression levels of miR-196b are restricted to MLL-rearranged leukemia cases, we measured the expression in samples of 72 selected pediatric acute lymphoblastic leukemia (ALL) cases i.e. MLL-rearranged and non-MLL-rearranged precursor B-ALL and T-ALL patients. MiR-196b was highly expressed in 9/12 MLL-rearranged precursor B-ALL patients, but also in 14/22 T-ALL patients. In particular, 100% of T-ALL cases carrying CALM-AF10 (n=5), MLL-AF6 (n=2), SET-NUP214 (n=3) and an inversion of chromosome 7 (n=1) showed high expression levels of miR-196b comparable to the high levels found in MLL-rearranged ALL. Like MLL-rearrangements, these genetic abnormalities have been functionally linked with upregulation of HOXA cluster genes. MiR-196b expression levels in these patients were strongly correlated with the expression of HOXA family but not with HOXB and HOXC cluster genes (Rs ≥ 0.7, P≤0.003). Since miR-196b is located between HOXA9 and HOXA10 on chromosome 7, our data suggest co-activation of miR-196b and HOXA family genes in pediatric ALL. In parallel to the high expression level of miR-196b we found decreased methylation at CpG islands located 5' of miR-196b in MLL-rearranged cases compared to normal bone marrow cells, which suggests an epigenetic origin for the high expression level of miR-196b in these patients. Despite the fact that MLL-rearranged ALL patients often respond poorly to prednisolone and L-asparaginase, upregulation of miR-196b was not indicative for the resistance to these drugs in pediatric ALL. In conclusion, high-level expression of miR-196b is not only MLL-driven, but can also be found in other types of leukemia that display aberrant activation of HOXA genes. Disclosures:No relevant conflicts of interest to declare.