Infant Leukemia Research Articles

Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia.

Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3D835H or NRASG13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

Diagnostic challenges in acute monoblastic/monocytic leukemia in children.

Acute monoblastic/monocytic leukemia (AMoL), previously defined as M5 according to FAB classification, is one of the most common subtypes of Acute Myeloid Leukemia (AML) in children, representing ~15–24% of all pediatric AMLs. Currently, the characterization of monocytic-lineage neoplasia at diagnosis includes cytomorphology, cytochemistry, immunophenotyping by multiparametric flow cytometry, cytogenetics, and molecular biology. Moreover, measurable residual disease (MRD) detection is critical in recognizing residual blasts refractory to chemotherapy. Nonetheless, diagnosis and MRD detection may still be challenging in pediatric AMoL since the morphological and immunophenotypic features of leukemic cells potentially overlap with those of normal mature monocytic compartment, as well as differential diagnosis can be troublesome, particularly with Juvenile Myelomonocytic Leukemia and reactive monocytosis in infants and young children. A failure or delay in diagnosis and inaccuracy in MRD assessment may worsen the AMoL prognosis. Therefore, improving diagnosis and monitoring techniques is mandatory to stratify and tailor therapies to the risk profile. This Mini Review aims to provide an updated revision of the scientific evidence on pediatric AMoL diagnostic tools.

A convergent malignant phenotype in B-cell acute lymphoblastic leukemia involving the splicing factor SRRM1.

A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion and that could potentially complement current clinical strategies in infant B-ALL.

Tisagenlecleucel therapy for relapsed or refractory B-cell acute lymphoblastic leukaemia in infants and children younger than 3 years of age at screening: an international, multicentre, retrospective cohort study

Children aged younger than 3 years were excluded from the ELIANA phase 2 trial of tisagenlecleucel in children with acute lymphoblastic leukaemia. The feasibility, safety, and activity of tisagenlecleucel have not been defined in this group, the majority of whom have high-risk (KMT2A-rearranged) infant acute lymphoblastic leukaemia and historically poor outcomes despite intensification of chemotherapy, and for whom novel therapies are urgently needed. We aimed to provide real-world outcome analysis of the feasibility, activity, and safety of tisagenlecleucel in younger children and infants with acute lymphoblastic leukaemia. We did an international, multicentre, retrospective cohort study at 15 hospitals across ten countries in Europe. Eligible patients were children aged younger than 3 years at screening between Sept 1, 2018, and Sept 1, 2021, who were screened for tisagenlecleucel therapy for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia according to licensed indications. Patients received a single intravenous infusion of tisagenlecleucel. We tracked chimeric antigen receptor T-cell therapy outcomes using a standardised data reporting form. Overall survival, event-free survival, stringent event-free survival, B-cell aplasia, and toxicity were assessed in all patients who received a tisagenlecleucel infusion. 38 eligible patients were screened, of whom 35 (92%) received a tisagenlecleucel infusion. 29 (76%) of 38 patients had KMT2A-rearranged acute lymphoblastic leukaemia, and 25 (66%) had relapsed after previous allogeneic haematopoietic stem-cell transplantation (HSCT). Patients had previously received a median of 2 lines (IQR 2-3) of (non-HSCT) therapy. Seven (18%) of 38 patients had received inotuzumab and 14 (37%) had received blinatumomab. After a median of 14 months (IQR 9-21) of follow-up, overall survival at 12 months after tisagenlecleucel infusion was 84% (64-93; five patients had died), event-free survival was 69% (47-83; nine events), and stringent event-free survival was 41% (23-58; 18 events). The probability of ongoing B-cell aplasia was 70% (95% CI 46-84; seven events) at 12 months. Adverse events included cytokine release syndrome, which occurred at any grade in 21 (60%) of 35 patients and at grade 3 or worse in five (14%), and neurotoxicity at any grade in nine (26%), none of which were severe. Measurable residual disease-negative complete response with or without haematological recovery occurred in 24 (86%) of 28 patients who had measurable disease. These data suggest that tisagenlecleucel has antitumour activity and has an acceptable safety profile for young children and infants with B-cell precursor acute lymphoblastic leukaemia. None.

Abstract 1075: Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate

Abstract Acute myeloid leukemia (AML) in infants is a unique subtype of AML that is driven by oncogenic fusions, resulting in a highly proliferative disease. The CBFA2T3-GLIS2 oncogenic fusion that is seen exclusively in infants and young children <3 years is the most refractory AML with near uniform mortality with conventional therapies. As part of our Target Pediatric AML (TpAML) discovery effort to define novel therapeutic targets, we discovered folate receptor alpha (FRα, FOLR1) transcript to be expressed in CBF-GLIS positive AML and verified cell surface expression of FOLR1 on leukemic cells. We present pre-clinical efficacy of a novel FOLR1 directed therapy (ELU001) in CBF-GLIS AML.ELU001 is a novel ~6 nm-diameter FOLR1-targeted C’Dot-Drug-Conjugate (CDC) incorporating ~21 exatecan molecules as a payload that is designed to treat cancers with improved safety and efficacy as compared to antibody-drug conjugates. A Phase 1 clinical trial of ELU001 in adult patients with tumors that overexpress FOLR1 is currently underway (NCT05001282). In this study, we determined the potential utility of ELU001 for CBFA2T3-GLIS2 AML by evaluating its preclinical efficacy in AML models. We confirmed the binding of ELU001 to AML cells expressing FOLR1 by flow cytometry using MV4;11 AML cell line transduced with an FOLR1 expression construct (MV4;11 FOLR1+). We show that MV4;11 FOLR1+ cells readily bind to ELU001 but not MV4;11 parental cells. Consistent with target-dependent binding, in vitro cytotoxicity studies demonstrated that MV4;11 FOLR1+ cells were highly susceptible to ELU001 (IC50=30 pM) while MV4;11 parental cells were only minimally affected by treatment. To evaluate in vivo efficacy, we transplanted MV4;11 FOLR1+ cells transduced with Luciferase-expression construct into immune deficient NSG mice at 1M cells per mouse and treated the mice the following week with different doses of ELU001 (vehicle, 0.3mg/kg Q3Dx6, 0.5 mg/kg Q3Dx3, and 0.65mg/kg Q3Dx3). Leukemia burden was assessed by whole-body bioluminescence (IVIS) imaging, performed weekly. IVIS imaging showed that ELU001 effectively eradicated the leukemia in vivoat all three doses tested resulting in enhanced survival while mice that received the vehicle treatment exhibited disease progression and eventually succumb to the disease. We confirmed that the leukemia was absent in ELU001-treated mice at the study endpoint of 120 days post treatment by flow cytometric analysis of the bone marrow, spleen and liver tissues harvested at necropsy. ELU001 was well tolerated with an apparent maximum tolerance dose of 1.50 mg/kg/mouse (0.5 mg/kg exatecan, Q3DX3). In summary, we demonstrated that ELU001 is highly effective at eliminating FOLR1 positive AML cells in vitro and in vivo, providing the preclinical data to support further assessment of ELU001 in clinical trials for pediatric patients with CBFA2T3-GLIS2 fusions overexpressing FOLR1. Citation Format: Quy Le, Feng Chen, Thao Tang, Cynthia Nourigat McKay, Fei Wu, Melik Turker, Thomas Gardinier, Vaibhav Patel, Aranapakam Venkatesan, Tin Khor, Kai Ma, Gregory P. Adams, Soheil Meshinchi. Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1075.

RARE-06. Expanding the clinical and molecular spectrum of pituitary blastoma

Pituitary blastomas (PitB) are rare DICER1-associated tumors that occur exclusively in children < 2 years of age. They are locally aggressive tumors that are driven by a combination of germline and somatic alterations involving DICER1. Here, we report two patients with pituitary neoplasms that expand the clinical and molecular spectrum of PitB. Patient 1 presented at 10 months with diabetes insipidus and was initially diagnosed with pituitary ependymoblastoma. She received debulking, chemotherapy and focal radiation with complete response achieved, but unfortunately died at the age of 8 years due to cerebral edema. Patient 2 was a survivor of infant leukemia who was treated with chemotherapy and then further chemotherapy with cranial irradiation at relapse. The patient was then diagnosed at the age of 8 years with pituitary CNS-PNET, which was treated with craniospinal irradiation and chemotherapy, and had remained in remission for 6 years. Review of histology in both cases indicates presence of neuroendocrine lobules, primitive cells, and Rathke pouch-like glandular structures, with high Ki67, ACTH and PRAME positivity compatible with PitB. Next-generation sequencing revealed presence of two DICER1 mutations (germline frameshifting and somatic missense) in Patient 1, and one somatic missense DICER1 mutation plus loss of heterozygosity in Patient 2 (no germline alteration). Surprisingly, C19MC amplification was also detected in Patient 1. Methylation profiling confirms clustering among our samples and PitB references, but not ETMR references. MicroRNA array revealed decrease in mature microRNA expression and preferential down-regulation of 5p/3p species in tumor compared to control pituitary tissue. In all, PitBs may present with clinical and molecular characteristics not conforming with the classical descriptions. It might be prudent to consider sequencing for DICER1 alteration in pediatric pituitary tumors to facilitate diagnosis of this increasingly heterogeneous rare entity.

Menin inhibitors as targeted therapeutics in KMT2a rearranged infant leukemia and the identification of effective treatment combinations.

10024 Background: KMT2a rearrangements are a hallmark of infant (less than 1 year of age) leukemia and are associated with poor prognosis. Menin is a ubiquitous protein that binds to the N-terminal of the KMT2a-fusion protein to mediate the oncogenic activity of KMT2a-rearranged leukemia cells. In this study, we evaluated the effect of multiple menin inhibitors and effective drug combinations for the treatment of KMT2A-rearranged infant leukemia. Methods: FISH analysis and immunoblotting confirmed the presence of KMT2a-fusion in the primary patient samples and cell lines studied. Lymphocytes from healthy donors were used as controls. Cells were treated with various concentration of multiple menin inhibitors for 72 hours and growth inhibition was measured using alamar blue assay. Infant leukemia cells were treated with a panel of FDA-approved agents (n = 221) to identify potential synergy, additive- and antagonistic-effects with specific menin inhibitors. Therapeutic drug interaction properties were established by calculating combination indices (CI) using the Chou-Talalay method. Mode of cell death and cellular target modulations were determined by western blot analysis. The effect of targeting menin in the context of the leukemogenic bone marrow microenvironment was determined through stromal cell co-cultures. Results: A comprehensive analysis of menin inhibitors on infant leukemia cell lines and primary patient cells exhibited significant and quantitatively diverse responses in cells with distinct molecular properties. Within the panel of menin inhibitors, infant B-ALL cells were found to be more sensitive to MI-463, MI-503 and MI-136 with a mean IC50 of 5.9µM, 6.1µM and 10.2µM, respectively. On the other hand, MI-3 exhibited an IC50 of 35.6µM. The cytotoxic effect of menin inhibitors on normal lymphocytes was minimal, suggesting a favourable therapeutic window (p &lt; 0.0001). High-throughput screening with a library of &gt; 200 FDA-approved drugs revealed significant sensitivity of distinct infant leukemia cells to proteasome, HDAC and CDK9 inhibitors. Among these, substantial drug synergy was observed between menin and proteasome inhibitors. For example, carfilzomib synergised with menin inhibitors over a broad range of concentrations with a CI value of 0.7. Conclusions: In this study, we present and discuss the initial proof-of-concept preclinical data for the effective anti-leukemic activity of menin inhibition against KMT2A-rearranged infant leukemia cells. Furthermore, the comprehensive drug screen and drug combination studies identified a spectrum of mechanistically-validated synergies, providing usable data for the formulation of multi-agent clinical studies for this currently unmet need in pediatric oncology.

Direct targeted therapy for MLL-fusion-driven high-risk acute leukaemias.

BackgroundImproving the poor prognosis of infant leukaemias remains an unmet clinical need. This disease is a prototypical fusion oncoprotein‐driven paediatric cancer, with MLL (KMT2A)‐fusions present in most cases. Direct targeting of these driving oncoproteins represents a unique therapeutic opportunity. This rationale led us to initiate a drug screening with the aim of discovering drugs that can block MLL‐fusion oncoproteins.MethodsA screen for inhibition of MLL‐fusion proteins was developed that overcomes the traditional limitations of targeting transcription factors. This luciferase reporter‐based screen, together with a secondary western blot screen, was used to prioritize compounds. We characterized the lead compound, disulfiram (DSF), based on its efficient ablation of MLL‐fusion proteins. The consequences of drug‐induced MLL‐fusion inhibition were confirmed by cell proliferation, colony formation, apoptosis assays, RT‐qPCR, in vivo assays, RNA‐seq and ChIP‐qPCR and ChIP‐seq analysis. All statistical tests were two‐sided.ResultsDrug‐induced inhibition of MLL‐fusion proteins by DSF resulted in a specific block of colony formation in MLL‐rearranged cells in vitro, induced differentiation and impeded leukaemia progression in vivo. Mechanistically, DSF abrogates MLL‐fusion protein binding to DNA, resulting in epigenetic changes and down‐regulation of leukaemic programmes setup by the MLL‐fusion protein.ConclusionDSF can directly inhibit MLL‐fusion proteins and demonstrate antitumour activity both in vitro and in vivo, providing, to our knowledge, the first evidence for a therapy that directly targets the initiating oncogenic MLL‐fusion protein.

MLL-AF4 and a murinized pSer-variant thereof are turning on the nucleolar stress pathway

BackgroundRecent pathomolecular studies on the MLL-AF4 fusion protein revealed that the murinized version of MLL-AF4, the MLL-Af4 fusion protein, was able to induce leukemia when expressed in murine or human hematopoietic stem/progenitor cells (Lin et al. in Cancer Cell 30:737–749, 2016). In parallel, a group from Japan demonstrated that the pSer domain of the AF4 protein, as well as the pSer domain of the MLL-AF4 fusion is able to bind the Pol I transcription factor complex SL1 (Okuda et al. in Nat Commun 6:8869, 2015). Here, we investigated the human MLL-AF4 and a pSer-murinized version thereof for their functional properties in mammalian cells. Gene expression profiling studies were complemented by intracellular localization studies and functional experiments concerning their biological activities in the nucleolus.ResultsBased on our results, we have to conclude that MLL-AF4 is predominantly localizing inside the nucleolus, thereby interfering with Pol I transcription and ribosome biogenesis. The murinized pSer-variant is localizing more to the nucleus, which may suggest a different biological behavior. Of note, AF4-MLL seems to cooperate at the molecular level with MLL-AF4 to steer target gene transcription, but not with the pSer-murinized version of it.ConclusionThis study provides new insights and a molecular explanation for the described differences between hMLL-hAF4 (not leukemogenic) and hMLL-mAf4 (leukemogenic). While the human pSer domain is able to efficiently recruit the SL1 transcription factor complex, the murine counterpart seems to be not. This has several consequences for our understanding of t(4;11) leukemia which is the most frequent leukemia in infants, childhood and adults suffering from MLL-r acute leukemia.

A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth

BackgroundChromosome translocations involving mixed lineage leukemia 1 (MLL1) cause acute leukemia in most infants and 5–10% children/adults with dismal clinical outcomes. Most frequent MLL1-fusion partners AF4/AFF4, AF9/ENL and ELL, together with CDK9/cyclin-T1, constitute super elongation complexes (SEC), which promote aberrant gene transcription, oncogenesis and maintenance of MLL1-rearranged (MLL1-r) leukemia. Notably, ENL, but not its paralog AF9, is essential for MLL1-r leukemia (and several other cancers) and therefore a drug target. Moreover, recurrent ENL mutations are found in Wilms tumor, the most common pediatric kidney cancer, and play critical roles in oncogenesis.MethodsProteolysis-Targeting Chimera (PROTAC) molecules were designed and synthesized to degrade ENL. Biological activities of these compounds were characterized in cell and mouse models of MLL1-r leukemia and other cancers.ResultsCompound 1 efficiently degraded ENL with DC50 of 37 nM and almost depleted it at ~ 500 nM in blood and solid tumor cells. AF9 (as well as other proteins in SEC) was not significantly decreased. Compound 1-mediated ENL reduction significantly suppressed malignant gene signatures, selectively inhibited cell proliferation of MLL1-r leukemia and Myc-driven cancer cells with EC50s as low as 320 nM, and induced cell differentiation and apoptosis. It exhibited significant antitumor activity in a mouse model of MLL1-r leukemia. Compound 1 can also degrade a mutant ENL in Wilms tumor and suppress its mediated gene transcription.ConclusionCompound 1 is a novel chemical probe for cellular and in vivo studies of ENL (including its oncogenic mutants) and a lead compound for further anticancer drug development.

Acute lymphoblastic leukemia (ALL) with KMT2A gene rearrangement in infants - characteristic clinical picture based on a case report

The most common malignancy affecting children is leukemia, which in infants refers to be diagnosed before 1 year of age and is relatively rare, but remains a problem for clinicians due to its aggressive clinical presentation, poor response to current treatments, and molecular biology. Infants with acute leukemia tend to present with aggressive features, including hyperleukocytosis, hepatosplenomegaly, central nervous system (CNS) involvement, and cutaneous infiltration. In infant leukemia, the KMT2A gene rearrangement at chromosome 11q23 is quite common. Infants diagnosed with acute leukemia harboring a 11q23 rearrangement have a particularly poor prognosis when compared to other children with acute leukemia.&#x0D; A 10-month-old girl was admitted in December 2017 to the Department of Pediatric Hematology, Oncology and Transplantology in Lublin with the suspicion of leukemia. One week before hospitalization minor bruises began to appear on the skin, unrelated to an injury. The child was admitted to the hospital, and there a complete blood count showed anemia, hiperleukocytosis and thorombocytopenia. On the admission to the Clinic: the severe general condition of the patient, with features of cardiopulmonary insufficiency were confirmed. Due to the patient's age and the presence of the KMT2A/MLLT3-t(9;11) (p22;q23) gene rearrangement, the infant was stratified as a high-risk group (HRG) and chemotherapy was started in accordance with the therapeutic program.&#x0D; Infant leukemia is one of the most difficult clinical situations encountered in pediatric oncology. Given the infant’s vulnerability and unique genetic rearrangements, there is a need to develop new protocols and therapies for infants.

A distinct hematopoietic differentiation state characterizes aggressive infant leukemia.

A distinct hematopoietic differentiation state characterizes aggressive infant leukemia.

High-Throughput Drug Library Screening in Primary KMT2A-Rearranged Infant ALL Cells Favors the Identification of Drug Candidates That Activate P53 Signaling.

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.

The Opportunity of Proteomics to Advance the Understanding of Intra- and Extracellular Regulation of Malignant Hematopoiesis.

Fetal and adult hematopoiesis are regulated by largely distinct sets of cell-intrinsic gene regulatory networks as well as extracellular cues in their respective microenvironment. These ontogeny-specific programs drive hematopoietic stem and progenitor cells (HSPCs) in fetus and adult to divergent susceptibility to initiation and progression of hematological malignancies, such as leukemia. Elucidating how leukemogenic hits disturb the intra- and extracellular programs in HSPCs along ontogeny will provide a better understanding of the causes for age-associated differences in malignant hematopoiesis and facilitate the improvement of strategies for prevention and treatment of pediatric and adult acute leukemia. Here, we review current knowledge of the intrinsic and extrinsic programs regulating normal and malignant hematopoiesis, with a particular focus on the differences between infant and adult acute leukemia. We discuss the recent advances in mass spectrometry-based proteomics and its opportunity for resolving the interplay of cell-intrinsic and niche-associated factors in regulating malignant hematopoiesis.

3105 – IMPACT OF DACH1 DOWNREGULATION ON MLL-AF4 INFANT LEUKEMOGENESIS

Leukaemia in infants, caused by a rearrangement of the MLL gene, has a very bad prognosis. The most common type of infant leukaemia is caused by the t(4;11) translocation that fuses the MLL and AF4 genes. Current lack of knowledge of its developmental origins and early stages of pathogenesis is one of the reasons for the slow progress in developing more targeted therapies. RNA-Seq studies have recently been completed in the lab to gain a more comprehensive insight into the molecular determinants that make foetal haematopoietic stem and progenitor cells more susceptible to transformation by MLL-AF4. A lack of expression of the DACH1 gene, which has been described as a tumour suppressor in a range of different cancers, was noted. The aim of this work is to assess the impact of DACH1 on the initiation of MLL-AF4+ infant leukaemia and on its maintenance. To determine the role of DACH1 on the initiation of leukaemia, we used our pre-leukaemia mouse model with Mll-AF4 expression in foetal cells and then transduced the cells with lentiviruses carrying a DACH1 overexpression construct. Using methylcellulose assays under B lymphoid or myeloid conditions, we observed that DACH1 reduces the size and numbers of B cell colonies and has a general negative effect on cell proliferation and differentiation. To establish whether DACH1 repression is required for leukaemia maintenance, we have overexpressed DACH1 in the SEM leukaemia cell line, which was derived from an MLL-AF4+ paediatric B-ALL. Our work shows that DACH1 affects negatively the proliferation and survival of the cells. To conclude, our data give new insights into the role of DACH1 in MLL-AF4 leukaemia. In the future, we will identify downstream targets of this protein to determine which genes are directly repressed by DACH1 to counteract the leukaemic phenotype.

Acute lymphoblastic leukemia in infants: A quarter century of nationwide efforts in Japan.

Acute lymphoblastic leukemia (ALL) with KMT2A gene rearrangement (KMT2A-r) in infants is a biologically and clinically unique disease and one of the most difficult to cure forms of pediatric leukemia. Multicenter clinical trials have been carried out in Japan since the mid-1990s by introducing allogeneic hematopoietic stem cell transplantation (HSCT) in first remission, which led to a modest improvement in outcome of infants with KMT2A-r ALL. Because of the emerging evidence that HSCT does not benefit every infant with KMT2A-r ALL, the Japanese Pediatric Leukemia/Lymphoma Study Group trial MLL-10 introduced risk stratification using age and presence of central nervous system leukemia, and introduced intensive chemotherapy, including high-dose cytarabine in early consolidation; indication of HSCT was restricted to the patients with high-risk features. The trial resulted in excellent 3-year event-free survival of 66.2% (standard error, 5.6%) and overall survival of 83.9% (standard error, 4.3%) for 75 patients with KMT2A-r ALL recruited between 2011 and 2015. This Japanese experience and the results of the infant ALL trials worldwide suggest the importance of introducing effective therapy in the early phase of therapy, thus clearing minimal residual disease as rapidly as possible. However, further improvement in outcome is unlikely with conventional treatment approaches. Introduction of biology-driven novel agents and/or immunotherapies through international collaboration would be key solutions to overcome the disease.

3093 – SUBTYPE-SPECIFIC INDUCTION OF MITOCHONDRIAL APOPTOSIS AS A NOVEL THERAPEUTIC OPTION IN HIGH-FATALITY PEDIATRIC LEUKEMIA

A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML. A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML.

Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants

▪MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified.We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof.Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM.To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising >200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models.In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro.Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. DisclosuresNo relevant conflicts of interest to declare.

SKIDA1 Sustains MLL-ENL-Expressing Hematopoietic Stem and Progenitor Cells

Chromosomal translocations involving the MLL1 gene often drive infant acute myeloid leukemia (AML). MLL fusion proteins (e.g., MLL-ENL, MLL-AF9, MLL-AF10) activate self-renewal programs in hematopoietic stem and progenitor cells, ultimately leading to transformation. The high frequency of MLL1 rearrangements in infant leukemias suggests that neonatal progenitors are uniquely poised to transform in response to these mutations. Indeed, we have recently shown that MLL-ENL initiates AML more efficiently in neonatal progenitors than in adult progenitors (Okeyo-Owuor et. al. Blood Advances, 2019). This raises the question of whether MLL-ENL induces key effectors of transformation more efficiently in neonatal progenitors than in adult progenitors. We identified Ski/Dach Domain Containing 1 (Skida1) as a gene that is highly induced by MLL-ENL in neonatal, but not adult hematopoietic progenitors. SKIDA1 is also highly expressed in human pediatric AML, and this expression is largely restricted to leukemias with MLL1 rearrangements. These observations suggest that Skida1 may be a critical, neonate-specific effector of MLL-ENL driven leukemogenesis.To test whether SKIDA1 plays a role in hematopoiesis and leukemogenesis, we generated a germline loss-of-function mouse allele. First, we tested whether loss of SKIDA1 perturbed hematopoiesis by flow cytometry. SKIDA1 deletion caused modest expansion of hematopoietic stem cells (HSCs) in neonates at post-natal day 0 (P0) and a modest decrease of hematopoietic committed progenitor cells (HPCs) at P14, but no changes in fetal or adult HSC/HPC numbers were observed. We then assessed HSC function by transplanting HSCs into lethally irradiated mice to assess their multi-lineage repopulating potential. Loss of SKIDA1 did not perturb HSC function at any stage of development, indicating a minor role in normal hematopoiesis. Next, we tested whether SKIDA1 modulates HSC or HPC numbers in the context of MLL-ENL expression. We crossed Skida1-/- mice to mice with a tetracycline inducible MLL-ENL transgene. The resulting mice (TetO-MLL-ENL; Vav1-Cre) express MLL-ENL specifically in hematopoietic cells beginning at embryonic day 10.5 (E10.5) in the absence of doxycycline, concordant with the onset of Vav1-Cre expression. When we induced MLL-ENL expression in Skida1 -/- mice, we observed near complete loss of HSCs and a severe reduction of HPCs. The reduction in HSC and HPC numbers in TetO-MLL-ENL; Vav1-Cre; Skida1 -/- neonates was far more severe than we observed in TetO-MLL-ENL; Vav1-Cre mice alone. Furthermore, this phenotype emerged between P0 and P14, indicating that it requires a neonatal context to manifest. This coincides with the developmental stage at which MLL-ENL most efficiently induces AML (Okeyo-Owuor et. al. Blood Advances, 2019). This data suggest that SKIDA1 helps sustain pre-leukemic MLL-ENL-expressing HSCs and HPCs shortly after birth. Ongoing studies will address the mechanism by which SKIDA1 regulates AML.Our current work raises two important points. First, SKIDA1 appears to have a far more important role in sustaining MLL-ENL-expressing progenitors than normal progenitors, raising the prospect for an excellent therapeutic window. Secondly, our findings show that age-specific MLL-ENL targets can shape pre-leukemic hematopoiesis and potentially AML initiation. This may help explain why MLL1 rearrangements account for a higher percentage of infant and childhood leukemias than adult leukemias. DisclosuresNo relevant conflicts of interest to declare.

Early Hematopoietic Stem Cell Transplantation in the Treatment of High-Risk Infant Leukemia

INTRODUCTIONAcute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are rare diseases in children less than one and two years old respectively. Infant ALL and AML demonstrates unique biological characteristics associated with aggressive clinical features, i.e. high frequency of KMT2A gene rearrangements, poor response to standard chemotherapy, and thus worse prognosis compared to older children. These patients (pts) are at high risk (HR). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for HR infant leukemia especially in 1 st CR, but proportion of these patients unable to undergo allo-HSCT due to early events, mainly relapse or treatment-related toxicity during initial therapy. We investigated the role of early allo-HSCT from different types of donors. The primary end points were overall survival (OS), transplant-relate mortality (TRM), graft-versus-host disease (GVHD) free, relapse-free survival (GRFS). Secondary end points included neutrophil engraftment and acute and chronic GVHD. MethodsWe included 50 infants with HR ALL (n=10, of with MLLr+ n=6) and AML (n=40, of with MLLr+ n=14) in CR 1. Median age at allo-HSCT 12,5 months old (range from 4 m.o. till 35 m.o.). Median time from diagnosis to allo-HSCT was 9 months (range from 3 till 21). Donor of SCT was: MSD-10% (n=5), MUD-46% (n=23), Haplo-44% (n=22). Majority of patients, 86% received MAC based on busulfan 12-16mg/kg (n=43, including IV busulfan-20pts), 14% (n=7) received RIC based on melphalan 140 mg/m 2. GVHD prophylaxis consisted of PTCy±CNI and m-TOR inhibitor in 62% (n=31), seroprophylaxis±CNI in 38% (n=19). Median follow-up was 64,3 months (range 1-220). Patient data were censored at the time of death or last follow-up. Probabilities of OS and GRFS were estimated using Kaplan-Meier curves. TRM was defined as any death that occurred in the absence of disease relapse; relapse was a competing risk for this event. ResultsEngraftment rate was 82%. Cumulative incidences by day 100 of TRM in Haplo were 2% vs 4% in MSD+MUD (р=0,9). Ten-year rates OS in Haplo and MSD+MUD were 77,3% (±4,7%) vs 78,6% (±9%) respectively (p=0,73). Ten-year GRFS did not differ between two study cohorts being 50% vs 57% for Haplo and MSD+MUD (p=0,76). Clinically significant aGVHD (grades 2-4) after allo-HSCT was observed in 27% (n=6) Haplo and 32% (n=9) MSD+MUD (p=0,77). A total of 12% (n = 6) patients developed severe cGVHD. ConclusionsEarly allogeneic HSCT in 1 st CR from different types of donors, including haploidentical is associated with favorable outcomes in infant acute leukemias. This approach should be evaluated in further controlled clinical studies. DisclosuresKulagin: X4 Pharmaceuticals, Alexion, Apellis, Biocad: Research Funding; Novartis, Generium, Sanofi, Roche, Johnson & Johnson, Pfizer: Speakers Bureau.