Infant Leukemia Research Articles

Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

▪MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood.We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression.In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. DisclosuresNo relevant conflicts of interest to declare.

Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Background:MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL.Methods:We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood.Results:We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR.To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect.Conclusions:MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infant leukaemia - faithful models, cell of origin and the niche.

ABSTRACTFor patients and their families, the diagnosis of infant leukaemia is devastating. This disease has not seen the improvements in outcomes experienced with other paediatric leukaemias and it is becoming ever more apparent that infant leukaemia is a distinct biological entity. Insights into some of the distinguishing features of infant leukaemia, such as a single mutation – the MLL-gene rearrangement, the biology of disease aggressiveness and lineage plasticity, and the high incidence of central nervous system involvement, are likely to be gained from understanding the interactions between leukaemic cells and their environment or niche. The origins of infant leukaemia lie in the embryonic haematopoietic system, which is characterised by shifting locations and dynamic changes in the microenvironment. Understanding this foetal or embryonic context is integral to understanding infant leukaemia development. Owing to its rarity and prenatal origins, developing accurate modelling systems for further investigation of infant leukaemia is essential. In this Review, we discuss how available in vitro, ex vivo and in vivo infant leukaemia models contribute to our current understanding of the leukaemia niche in embryonic development, established disease and specialised non-haematopoietic niches. The mechanistic insights provided by accurate models will help identify viable novel therapeutic options.

Defining the fetal origin of MLL-AF4 infant leukemia highlights specific fatty acid requirements.

SummaryInfant MLL-AF4-driven acute lymphoblastic leukemia (ALL) is a devastating disease with dismal prognosis. A lack of understanding of the unique biology of this disease, particularly its prenatal origin, has hindered improvement of survival. We perform multiple RNA sequencing experiments on fetal, neonatal, and adult hematopoietic stem and progenitor cells from human and mouse. This allows definition of a conserved fetal transcriptional signature characterized by a prominent proliferative and oncogenic nature that persists in infant ALL blasts. From this signature, we identify a number of genes in functional validation studies that are critical for survival of MLL-AF4+ ALL cells. Of particular interest are PLK1 because of the readily available inhibitor and ELOVL1, which highlights altered fatty acid metabolism as a feature of infant ALL. We identify which aspects of the disease are residues of its fetal origin and potential disease vulnerabilities.

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification

ABSTRACT Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

Infant T‐cell acute lymphoblastic leukaemia with t(6;7) (TCRB‐MYB) translocation

T-ALL is rare in infancy with only 10 (1.5%) of 651 patients of that subtype in the Interfant-06 infant ALL trial. We report 3 cases of t(6;7) (TCR/MYB) infant T-cell Acute Lymphoblastic Leukaemia who appear to have a distinct clinical presentation with CNS disease and refractory disease or late relapse.

Fetal and Neonatal Hematopoietic Progenitors Are Exquisitely Sensitive to MLL-ENL-Driven Leukemia Initiation

Translocations involving the MLL1/KMT2A gene are observed in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), but the frequency at which these mutations occurs is different at different stages of life. For example, MLL rearrangements are extremely common in infant leukemia (~70% of cases), but they are much less common in adolescent or adult AML (~5% of de novo adult AML). While the function of MLL fusion proteins has been extensively studied, it remains unclear why MLL-rearrangements are so common in infant leukemias as compared to adolescent or adult leukemias. One possibility is that fetal hematopoietic progenitors are more readily transformed by MLL fusion proteins than adult progenitors. Recent work in our lab has shown that a mutation commonly associated with adult AML, the FLT3-Internal Tandem Duplication, selectively activates leukemogenic transcriptional programs in adult as compared to fetal/neonatal progenitors (eLife 5:e18882). We have developed a system to test whether an analogous effect holds for MLL translocations - i.e. do they preferentially initiate leukemogenesis in fetal progenitors. We made use of a previously described tet-O doxycycline inducible mouse allele (Cell Reports 9:1246) to both spatially and temporally control expression of MLL-ENL, a common MLL fusion product (Fig. 1A). We found that MLL-ENL induction at fetal (E10.5) or neonatal (P0) stages caused AML to develop in most mice, but MLL-ENL induction at juvenile (P21) or adult (8 weeks) stages did not (Fig. 1B). Remarkably, AML developed with the greatest penetrance (100%) when MLL-ENL was induced at P0 as compared to both earlier and later developmental stages. In neonates, MLL-ENL profoundly depleted hematopoietic stem cells (HSCs) and, to a lesser extent, lineage-restricted hematopoietic progenitors (HPCs), and it caused expansion of myeloid progenitor populations. These effects began to dissipate by 10 weeks after birth. Changes in HSC and HPC frequency were not at all evident when MLL-ENL was induced at an adult stage, but by a year after MLL-ENL induction, the surviving mice had a significantly expanded HSC pool. MLL-ENL induced widespread changes in gene expression in neonatal HPCs. In contrast, very few changes in gene expression were observed in MLL-ENL expressing adult HPCs. Known targets of MLL-ENL, such as HoxA9, were more highly induced in neonatal HPCs than in adult HPCs, though expression was observed in older mice. Altogether, our data demonstrate a dynamic relationship between developmental stage and the transcriptional response to MLL-ENL, and they may help account for the high percentage of MLL rearrangements observed in infant leukemia. Furthermore, our findings have opened new lines of investigation into the relationship between the epigenetic landscapes of developing HSCs/HPCs and the programs that drive leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Preliminary Results of UCART19, an Allogeneic Anti-CD19 CAR T-Cell Product in a First-in-Human Trial (PALL) in Pediatric Patients with CD19+ Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

▪BackgroundUCART19 is a genetically modified T-cell product manufactured from non-HLA matched healthy donor cells and aims to provide a ready to use, off-the-shelf allogeneic engineered CAR T-cell product. Lentiviral-transduced CAR T-cells express (1) an anti-CD19 CAR (anti-CD19 scFv- 41BB- CD3ζ), and (2) an RQR8 “safety switch” that is intended to allow targeted elimination of RQR8+ cells by rituximab. UCART19 has been additionally modified to disrupt the T-cell receptor alpha constant (TRAC) and CD52 genes. We previously reported the success of this approach in two infants treated under compassionate use who achieved molecular remissions ahead of allogeneic transplantation and remain in remission with follow-up for 18 and 24 months. Here we describe preliminary results of a Phase 1 trial of UCART19 in pediatric patients (pts) with high risk relapsed refractory (R/R) CD19+ B-ALL and no therapeutic options.MethodsThis study evaluates the safety and tolerability of a fixed dose (2x107 total cells or 1.1 to 2.3x106 cells/kg) of UCART19 and its ability to achieve molecular remission at day (D) 28 to enable allogeneic stem cell transplantation (allo-SCT). Up to 10 subjects with R/R B-ALL aged between 6 months and < 18 years, with morphologically confirmed CD19+ B-ALL or an MRD load ≥ 1x10-3 are eligible. The lymphodepletion regimen combines cyclophosphamide and fludarabine with or without alemtuzumab (FC or FCA). UCART19 is administered thereafter (Day 0) as a single dose infusion. If the patient achieves molecular remission, allo-SCT is scheduled within 6 to 12 weeks (wks) after infusion, and trial period continues for a further 12 months before entry into a long-term follow-up study.ResultsAs of 24 June 2017, 5 children (3 males and 2 females), including two infant leukaemias, aged between 8 months and 16.4 years had been enrolled and treated. Four pts had received 3 or more previous lines of treatment and 2 had undergone a previous allo-SCT. All patients were unsuitable for or had failed generation of an autologous CAR T-cell product. All had received debulking chemotherapy and/or inotuzumab ozogamicin therapy before UCART19. All pts received lymphodepletion with FCA. No immediate infusion related reaction (IRR) related to UCART19 were reported. All pts experienced reversible cytokine release syndrome (CRS) (1 grade (G) 1, 3 G2, 1 G3), with one child requiring 2 doses of tocilizumab. Time to onset of first CRS symptoms was between D4 and D8. Two pts presented mild neurological symptoms that recovered (within 1 to 3 days) without treatment. Acute skin GvHD G1 was reported in two pts (one with biopsy confirmation) and both recovered with topical steroids. Four children experienced viral complications (CMV, ADV, BK, metapneumovirus) related to lymphodepletion, and all experienced neutropenia, which was prolonged in 2/5 pts. By D28-42, 5/5 pts had achieved complete remission with incomplete blood count recovery albeit with hypoplastic marrows (all patients were MRD negative by flow cytometry or qPCR ). UCART19 were detectable from D7 to at least D28 in all subjects by qPCR and/or flow cytometry and by molecular signatures of T-cell donor chimerism. All proceeded to conditioned allo-SCT between 7 to 9 weeks after UCART19 infusion, with no gene-modified cells detectable thereafter. Two children relapsed 3 months after transplantation (one CD19- and one CD19+) and died 7 and 8 months after UCART19 infusion respectively. One subject died in remission from transplant related complications including thrombotic microangiopathy and BK haemorrhagic cystitis and nephritis. Two subjects remain in molecular remission 2 and 2.5 months post-transplant.ConclusionPreliminary results of this first-in-human trial of UCART19 treatment in a high risk R/R B-ALL pediatric population revealed no unexpected toxicities. GvHD seen in 2 patients was mild and self-limiting. Lymphodepletion-related viral complications and persisting neutropenia were encountered. All five treated subjects were rendered either flow or PCR MRD negative enabling allogeneic transplantation to proceed. The trial is ongoing in the UK and further data may be presented (NCT02808442) . DisclosuresQasim:Autolus Ltd: Consultancy, Equity Ownership; Miltenyi: Research Funding; Orchard Therapeutics: Consultancy, Equity Ownership; Servier: Research Funding; Bellicum: Research Funding; NIHR: Research Funding; Cellectis: Research Funding. Lucchini:Alexion: Membership on an entity's Board of Directors or advisory committees. Zinaï:Servier: Employment. Binlich:Servier: Employment. Dupouy:Servier: Employment. Pauly:Servier: Employment. Balandraud:Servier: Employment. Dubois:Servier: Employment. Konto:Bristol-Myers Squibb: Employment, Equity Ownership; Pfizer: Employment, Equity Ownership. Patel:Pfizer: Employment, Equity Ownership. Veys:Bellicum: Research Funding; Servier: Research Funding.

Targeting the DNA Damage Response Pathway in MLL- rearranged B-Acute Lymphoblastic Leukemias

Infant B-acute lymphoblastic leukemias (B-ALL) that harbor MLL-AF4 rearrangements are associated with a poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL - r ) childhood ALL (EFS > 85%). Thus, effective therapies that ensure long-term durable responses are of extreme interest. Recently, the targeting of the DNA damage response pathway (DDR) has been shown to be cytotoxic to MLL-r acute myeloid leukemia (AML) cells with oncogene driven replicative stress. In order to determine if targeting of the DDR pathway would be efficacious in MLL-r B-ALLs, we tested a panel of human AML and ALL cell lines and found that all B-ALL cell lines (those with and without MLL-r ) were highly sensitive to ATR kinase inhibition. At 72 hours, IC50s for MLL- r B-ALL cell lines (RS4;11, SEMK2, Hb11;19) were 2-3 fold lower than MLL-AF9 AML cell lines (Molm13, THP-1, Nomo1). Inhibition of ATR kinase induced double stranded DNA damage (as assessed by the increase in phospho-γH2AX levels), apoptosis and loss of the G2-M cell cycle checkpoint in a dose dependent manner in all cells treated, with B-ALL cells exhibiting these effects more rapidly and at lower doses.In order to better understand the mechanism behind the increased sensitivity of MLL -r B-ALLs to ATR inhibition, we generated a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in MLL-Af4 murine model. Recipient mice injected with Mll-Af4/N-RasG12D pre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable, with accelerated latency to secondary and tertiary disease. Multiple sequencing studies have indicated that while MLL -r B-ALL have relatively few additional somatic mutations, activating mutants of RAS seem to co-occur at a frequency close to 50%. Moreover, among MLL -r infant leukemias, those harboring mutant RAS mutations have even worse overall prognosis, further emphasizing the utility of our model.We were able to detect increased basal levels of DNA damage and replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to a small molecule inhibitor of ATR, AZ20. In vitro and in vivo treatment with AZ20 resulted in increased leukemia cytotoxicity. However, tertiary B-ALL transplanted mice eventually succumbed to disease following prolonged AZ20 treatment in vivo . As our model includes an activating mutation in N-Ras , we hypothesized that cells would be sensitive to the combination with small molecule inhibitors of downstream effectors of Ras. The combination of AZ20 and MEK inhibitor GSK212 (Trametinib) increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivo combination treatment of not only our mouse model but also of patient derived xenografts, harboring both mutations, exhibited decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. These data suggest a potent combination therapy for the treatment of this subgroup of poor prognosis MLL-AF4 patients with RAS mutations.To try to gain some further mechanistic insight, we conducted gene set enrichment analysis of RNA-sequencing of primary Mll-Af4/N-RasG12D leukemias and normal pro-B cells. Our analyses revealed significant negative correlations with gene signatures involving the G2/M checkpoint and ATR activation in response to replication stress, suggesting derangements in DDR pathways. We validated these observations with quantitative RT-PCR of ATR pathway members in murine mouse models of MLL-r AML and B-ALL. Our studies suggest that the observed sensitivity to ATR inhibition correlates to the overall expression level of ATR and ATR pathway members. We observed similar patterns of sensitivity and expression in the human cell lines we tested, as well as in an analysis of published gene expression data of infant and pediatric B-ALL patients. Altogether, our data indicates the inhibition of ATR in cells with oncogene driven replicative stress and abrogated ATR and ATR pathway expression, as part of an effective combination therapeutic strategy in this subtype of acute leukemia. DisclosuresArmstrong:C4 Therapeutics: Consultancy; Imago: Consultancy; Epizyme: Consultancy; Cyteir: Consultancy.

Irinotecan Induces Complete Remission in MLL -Rearranged Acute Lymphoblastic Leukemia Xenografts

BACKGROUND: Acute lymphoblastic leukemia (ALL) in infants (diagnosis < 1 year) is characterized by a high incidence of rearrangements of the MLL (KMT2A ) gene, occurring in ~80% of the cases. The resulting MLL fusions drive a leukemia biologically and clinically distinct from other subtypes. Therefore, while current ALL treatments achieve cure rates of 80-90%, MLL -rearranged (MLL -R) infant ALL is still beset by poor outcome, with a 5-year event-free survival of ~40%. Further intensification of current treatment is not possible, highlighting an urgent need for new therapeutic strategies.AIM: While novel drug classes have shown pre-clinical promise in MLL -R ALL, clinical implementation has become a major bottle-neck, as set-up of clinical trials with new drugs, as well as their FDA-approval, is a protracted process. Hence, we explored a drug repurposing approach, performing screens of >3000 FDA-approved drugs to identify compounds able to both target MLL -RALL cells and expeditiously transition into clinics.METHODS:MLL -RALL cell lines (SEM, KOPN8 and RS411) were screened using commercially available drug libraries comprising in total 3685 compounds; drug sensitivity to the initial concentration of 1 μM was assessed using 4-day MTS-assays. The most efficacious hits were further screened at 100 nM and 10 nM, to refine selection of potent candidate compounds. Among the top hits were the topoisomerase I inhibitor camptothecin and its derivatives, 10-hydroxycamptothecin and 7-ethyl-10-hydroxycamptothecin (SN38). The most potent derivative, SN38, was further analyzed in vitro to assess SN38-induced molecular changes in MLL -RALL cells using immunoblotting and cell death assays.To assess the anti-leukemic effects of SN38 in vivo , immunodeficient NOD.Cg- PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were injected with the luciferase -expressing MLL -RALL reporter cell line SEM-SLIEW, as well as primary leukemic cells of two MLL -Rinfant ALL patients. Mice were treated 3 times per week with either the pro-dug irinotecan (40 mg/kg), which is metabolised into SN38, or vehicle, for up to 4 weeks. Treatment was initiated after engraftment was confirmed by either bioluminescence imaging (SEM-SLIEW) or presence of >1% human leukemic cells in the blood was detected by multi-color flow cytometry (patient-derived xenografts, PDX). Disease progression over time was measured accordingly. To investigate the curative potential of irinotecan, treatment with the drug was initiated in a subset of mice displaying advanced, full-blown leukemia.At the end of treatment, mice were killed to assess the effect of irinotecan on human leukemic cell engraftment in bone marrow (BM), blood and spleen using flow cytometry and histochemistry. In addition, a subset of irinotecan-treated mice was kept alive for up to 42 days without treatment, to monitor for disease relapse.RESULTS: Camptothecins target MLL -RALL cells in vitro ; in particular SN38 had nanomolar IC50-values in both cell lines and primary leukemicpatient cells. Exposure with 25 nM SN38 inhibited proliferation and induced cell death of the MLL -RALL cell line SEM; characteristic DNA damage markers, such as accumulation of γ-H2AX and CHK2 phosphorylation could also be detected already after 2h of SN38 exposure. The anti-leukemic effect of SN38 could be validated in vivo in MLL -RALL xenografts, where the SN38 pro-drug irinotecan completely blocked leukemia expansion. At the experimental end-point, flow cytometric analyses showed human leukemic cells to be completely eradicated from the BM, blood and spleen in irinotecan-treated cell line xenografts, and to be sporadically present at minimal residual disease levels in irinotecan-treated PDX mice. Histochemistry stainings confirmed lack of leukemic infiltration in extramedullary tissues. Moreover, irinotecan induced complete remission in mice with advanced leukemia, as shown by flow cytometry as well as loss of bioluminescence. While ablation of human leukemic cells persisted in cell line-derived xenografts after treatment stop, PDX mice relapsed after 4 weeks, possibly due to presence of leukemic cells at low levels; this could possibly be improved by extending treatment duration.CONCLUSION: Irinotecan inhibits MLL -R ALL very potently, and as an FDA-approved drug already used in other pediatric cancers, it is an ideal candidate for repurposing and implementation in future MLL -R ALL treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic and Transcriptomic Profiling of MLL Rearranged Infant Acute Lymphoblastic Leukemia

[Background] Infant acute lymphoblastic leukemia (ALL) accounts for less than 5% of childhood leukemia, and is characterized by Mixed lineage leukemia (MLL also called KMT2A ) rearrangements found in approximately 80% of patients. Also found in 5-10% of ALL in older children and adults, MLL -rearrangement positivity is highly associated with poor outcome in ALL; there is an urgent need for molecular findings of potential therapeutic targets and/or risk stratification markers, which can be translated into the clinical practice. Recently, the mechanisms that MLL fusion proteins recruit epigenetic regulators, including histone H3 lysine 79 methyltransferase DOT1L and BET family histone readers, to activate the transcription of target genes have been reported, suggesting epigenetic dysregulation should play a central role in the leukemogenesis. However, DNA-level methylation profiles of MLL -rearranged leukemia and their associations with gene expression profiles are not fully characterized.[Materials and Methods] Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. Both methylation array and RNA sequencing were performed on the diagnostic leukemia samples from 38 infants with ALL, consisting of 30 cases with MLL -rearrangements identified by fluorescence in situ hybridization or Southern blotting, and 8 MLL -germline cases. Methylation beta values were normalized with beta-mixture quantile normalization method. For RNA sequencing data analysis, we used publicly available Genomon Pipeline.[Results] RNA sequencing confirmed MLL -rearrangements in 30 cases, whose partner genes comprised AFF1 (n=15), MLLT1 (n=6), MLLT3 (n=3), MLLT10 (n=2), EPS15 (n=1), AFF2 (n=1), ABI1 (n=1), and SARNP (n=1). As expected, DNA methylation-based hierarchical clustering divided the cases into 2 clusters with highly distinctive methylation profiles: MLL -rearranged cluster (MLL -R cluster) and MLL -germline cluster (MLL -G cluster). Intriguingly, 3 out of 30 (10%) MLL -rearranged cases were classified into MLL -G cluster; these cases revealed uncommon partner genes, such as ABI1 , SARNP , and MLLT10, respectively. ABI1, an Eps8 SH3 domain-binding protein, and SARNP, a SAP domain containing ribonucleoprotein, are rare fusion partners of MLL. The MLL -germline leukemia-like methylation profiles suggested different leukemogenic mechanisms from common fusion partners, such as AFF1 and MLLT1, which directly or indirectly interact with DOT1L and positive transcription elongation factor b. Because DOT1L-binding domain of MLLT10 was retained in both of the 2 cases with MLL-MLLT10 in our cohort, additional mechanisms causing MLL-germline leukemia-like methylation in a single case may be present. As a result of inter-cluster comparison, MLL -R clusterexhibited global hypermethylation compared to MLL -G cluster (P -value < 2.2 × 10−16, Kolmogorov-Smirnov test). Methylation-based gene set enrichment analysis demonstrated significant promoter hypermethylation on cell development/differentiation-related gene sets and hypomethylation on DNA replication/cell proliferation-related gene sets (P -value < 0.01, hypergeometric test) in MLL -G cluster. Consistently, RNA sequencing-based gene expression analysis demonstrated overexpression of cell cycle regulators, such as cyclins/cyclin-dependent kinases, in MLL -R cluster, which may be associated with more aggressively proliferating diseases.[Conclusion] Corresponding to known aberrant histone modifications in MLL -rearranged leukemia, largely different DNA methylation profiles between MLL -R and MLL -G clusters have been demonstrated. Because DNA-level methylation on CpG dinucleotides interactively affects histone modifications, these results implicated aberrant DNA methylation in the pathogenesis of MLL -rearranged leukemia as a part of epigenetic dysregulation. We also identified a subset of MLL -rearranged cases with MLL -germline leukemia-like methylation/expression profiles, indicating that not only MLL -rearrangement positivity, but also the types of MLL -rearrangement partners may affect a biological feature of leukemic cells. These cases may possibly be therapeutically stratified as relatively lower risk group compared to other MLL -rearranged cases. Further detailed analyses using clinical information are planned. DisclosuresNo relevant conflicts of interest to declare.

CCG 1953: Acute Lymphoblastic Leukemia in Infants: Analysis of Prognostic Factors: A Report from the Children's Oncology Group.

Background: Infant ALL has a poor therapeutic outcome. In contrast to other subtypes of ALL, infants have not benefited from risk-adapted therapy and intensification of therapy. This is the first cooperative group trial characterizing all infants at the molecular level for MLL/11q23 rearrangements for better stratification in future trials.Procedure: All infants enrolled on CCG 1953 were tested for MLL rearrangement by Southern Blot, and the 11q23 translocation partner was identified (4;11, 9;11, 11;19, or “other”) by reverse-transcriptase PCR.Results: 115 infants were enrolled (July 1996 to August 2000). Treatment for patients consisted of 4 courses of induction-consolidation-intensification followed by 3 cycles of intensified maintenance and 4 cycles of routine maintenance. Infants with 11q23/MLL abnormalities and a protocol-defined 5-6/6 related or unrelated donor identified within 4 months were assigned to BMT. Central nervous system prophylaxis consisted of triple intrathecal therapy during the first 10 weeks of therapy. Infants not undergoing BMT received additional CNS prophylaxis with very high dose methotrexate. Patients then received one year of intensified maintenance, followed by a year of standard maintenance. Overall event-free survival (EFS) was 41.7% (SD = 9.2%) and overall survival (OS) was 44.8% at 5 years. Five-year EFS for MLL rearranged cases was 33.6%, and for MLL non-rearranged cases was 60.3% (p=.006). 5-year EFS for MLL/11q23 subgroups was as follows: t(4;11): 29.0%; t(11;19): 30.0%; t(9;11): 22.2%; and “other 11q23” 53.5% (p=.122). Multivariate Cox regression analyses showed a rank order of significance for negative impact on prognosis for CD10 negativity, age less than 6 months, and MLL rearrangement, in that order. We did not observe a statistically significant difference in outcome between different MLL translocation partners, though we did observe a non-significant trend toward improved outcome in patients with “other” MLL translocation partners as compared to those with t(4;11), t(9;11) or t(11;19). Toxicity was the most frequent cause of death. Relapse as a first event in 1953 was later (median 295 days) compared to the predecessor1883 infant ALL trial (median 207 days).Conclusions: MLL/11q23 rearrangement, CD10 expression, and age are important prognostic factors in infant ALL, but individual molecular 11q23 translocation partners do not predict outcome. These results indicate that certain subgroups have an unusually poor outcome and are therefore candidates for novel therapeutic approaches in first remission. Given the high mortality, further dose intensification strategies are unlikely to result in better long-term survival.

Irinotecan Induces Disease Remission in Xenograft Mouse Models of Pediatric MLL-Rearranged Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.

Blinatumomab following haematopoietic stem cell transplantation - a novel approach for the treatment of acute lymphoblastic leukaemia in infants.

Blinatumomab with subsequent haematopoietic stem cell transplantation was applied in 13 infants with acute lymphoblastic leukaemia (ALL). Eight patients were treated in first remission due to slow clearance of minimal residual disease (MRD); one for MRD-reappearance after long MRD negativity, one for primary refractory disease and three during relapse treatment. In slow MRD responders, complete MRD response was achieved prior to transplantation, with an 18-month event-free survival of 75%. In contrast, only one of five patients with relapsed/refractory ALL is still in complete remission. These data provide a basis for future studies of immunotherapy in very high-risk infant ALL.

P0699 / #555: LEUKEMIA IN A 1-MONTH-OLD FEMALE PRESENTING WITH ABDOMINAL DISTENSION AND CONSTIPATION

Aims & Objectives: Purpose: Acording to the literature pediatric leukemia is frequently presented in children between 1-10 years (60%) and there are a few cases in infants (less tan 4.7%). This is a case of an infant presenting with hyperleukocytosis, trombocitopenia and anemia who was diagnosed of acute lymphoblastic leukemia. Methods: Case Report: 1-month-old female presented to an outside hospital with abdominal distension, constipation, petechia and melena of 3 weeks of evolution, it was treated with formula change and laxative without improvement. A blood citometry was requested, 330,000 leukocytes and 17,000 platelets were reported. She was transferred to our hospital. Initial vital signs showed: temperature 37.2° C, heart rate 173 bpm, blood pressure 90/60 mmHg, oxygen saturation of 98%. An arterial blood gas showed metabolic acidosis, hypokalemia and hypocalcemia. Chest radiograph was normal. The patient recieved hyperhidratation intravenous fluids, oxygen via nasal canula and platelet transfusion and was admitted to the PICU. Results: Evolution: 24 hours admission: the patient present acute respiratory failure that requires mechanical ventilation, with persistent hypoxemia (pAFi: 63) that was treated with lung protection maneuvers and pronation for 14 hours, the hypoxemia improved after 3 days and ventilatory settings were reduced. 48 hours after admission the patient developed tumoral lysis syndrome (hyperuricemia, hyperphosphatemia and hypocalcemia), continuous renal replacement was started for 16 hours remitting tumoral lysis syndrome. Diagnosis of leukemia was confirmed and dexametasone was started at 5 mgm2scd. Conclusions: Discussion: Acute leukemia in infants is rare and usually this patients have a poor or fatal outcome.

Infant Acute Myeloid Leukemia: A Unique Clinical and Biological Entity.

Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children <2 years of age. It is as frequent as infant acute lymphoblastic leukemia (ALL) but not clearly distinguished by study groups. However, infant AML demonstrates peculiar clinical and biological characteristics, and its prognosis differs from AML in older children. Acute megakaryoblastic leukemia (AMKL) is very frequent in this age group and has raised growing interest. Thus, AMKL is a dominant topic in this review. Recent genomic sequencing has contributed to our understanding of infant AML. These data demonstrated striking features of infant AML: fusion genes are able to induce AML transformation without additional cooperation, and unlike AML in older age groups there is a paucity of associated mutations. Mice modeling of these fusions showed the essential role of ontogeny in the infant leukemia phenotype compared to older children and adults. Understanding leukemogenesis may help in developing new targeted treatments to improve outcomes that are often very poor in this age group. A specific diagnostic and therapeutic approach for this age group should be investigated.

Acute Leukemia in Infants.

Infant leukemia is a rare, distinct subgroup of pediatric acute leukemias diagnosed in children under 1year of age and characterized by unique, aggressive biology. Here, we review its clinical presentation, underlying molecular biology, current treatment strategies, and novel therapeutic approaches. Infant leukemias are associated with high-risk molecular features and high rates of chemotherapy resistance. International collaborative clinical trials have led to better understanding of the underlying molecular biology, refined risk-based stratification, and investigated the use of hematopoietic stem cell transplantation. However, intensification of chemotherapy has failed to improve outcomes, and current regimens are associated with significant treatment-related and long-term toxicities. Infants with leukemia remain a challenging group to treat. We must continue collaborative efforts to move beyond traditional cytotoxic chemotherapy, incorporate molecularly targeted strategies and immunotherapy, and increase access to clinical trials to improve outcomes for this high-risk group of patients.

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

Simple SummaryMLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex.MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

Acute leukemia of infants and neonates

Leukemias diagnosed in <1-year-old infants generally have an aggressive clinical nature and unique biological characteristics. Acute lymphoblastic leukemia (ALL) in infants is still intractable and difficult to treat as compared with other pediatric ALLs, for which considerable progress in treatment outcomes has been recently achieved. Infant leukemia cells frequently carry chromosome translocations involving the 11q23 locus, resulting in the rearrangement and fusion of the KMT2A (MLL) gene. Among several KMT2A fusion genes, KMT2A-AFF1 (MLL-AF4) fusion is characteristically observed in neonatal and infant ALL, representing a hallmark of poor prognosis. The cytogenetic/molecular abnormalities t (1;22)(p13.3;q13.1)/RBM15-MKL1 and t (8;16)(p11.2;p13.3)/KAT6A-CREBBP (MOZ-CBP) are also well-known in acute myeloblastic leukemia in this population. Although many neonatal leukemias occurring within the first 28 days of birth are refractory, spontaneous remissions are occasionally observed, especially in the case of t (8;16). Therefore, international collaborative studies are necessary to improve understanding and facilitate the development of better treatment for this rare disease. Thus, this study summarizes the recently reported clinical, cytogenetic, and molecular biology aspects of neonatal and infant leukemias.