Infant Acute Leukemia Research Articles

The role of LEDGF in transcription is exploited by HIV-1 to position integration.

HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.

Maternal diet in pregnancy and acute leukemia in infants: a case-control study in Mexico City.

Epidemiological studies around the world on acute leukemia (AL) and risk factors in infants are scarce. Infant AL has been proposed to originate in utero, which facilitates its study by establishing a short exposure time in pregnant women to environmental and dietary factors that could contribute to the risk of or protection against leukemia. We hypothesized that maternal diet during pregnancy may be an important factor involved in AL in offspring. We conducted a hospital-based case-control study from 2010 to 2019 on maternal diet during pregnancy in nine high-specialty public hospitals of different health institutions that diagnose and offer treatment to children with AL in Mexico City. Cases (n=109) were children ≤24 months of age with de novo diagnosis of AL, and controls (n=252) were children obtained in hospitals from second-level medical care matched for age, sex, and health institution. Maternal diet during pregnancy was obtained by a semiquantitative food frequency questionnaire. Unconditional logistic regression models were used to assess the association between food groups and infant AL. Potential confounders were assessed by constructing directed acyclic graphs (DAGs) with Dagitty software in which adjusted options were identified for the construction of unconditional logistic regression models. Cases were slightly predominantly female (52.3%). The years of education of the mother in cases and controls was 0-9 on average, and those who reported smoking cigarettes and consuming alcohol during pregnancy did so at a low frequency. Regarding the mother's diet, the main findings were that the consumption of allium vegetables during pregnancy was inversely associated with AL for medium and high consumption (OR=0.26, 95% CI 0.14-0.46; P-trend< 0.001). In contrast, the high consumption of high-fat dairy products had a positive association with AL (OR=2.37, 95% CI 1.30-4.34; P-trend<0.001). No association was found between consumption of topoisomerase II inhibitor foods during pregnancy and AL. The results suggest that maternal intake during pregnancy of allium vegetables, specifically garlic, is inversely associated with the development of AL in children ≤24 months old. On the other hand, consumption of high-fat dairy products is positively associated with AL in children ≤24 months old.

Maternal dietary patterns and acute leukemia in infants: results from a case control study in Mexico.

Childhood cancer is the leading cause of disease-related mortality among children aged 5-14 years in Mexico, with acute leukemia being the most common cancer among infants. Examining the overall dietary patterns allows for a comprehensive assessment of food and nutrient consumption, providing a more predictive measure of disease risk than individual foods or nutrients. This study aims to evaluate the association between maternal dietary patterns during pregnancy and the risk of acute leukemia in Mexican infants. A hospital-based case-control study was conducted, comparing 109 confirmed acute leukemia cases with 152 age-matched controls. All participants (≤24 months) were identified at hospitals in Mexico City between 2010 and 2019. Data on a posteriori dietary patterns and other relevant variables were collected through structured interviews and dietary questionnaires. Multivariate logistic regression was employed to estimate the association between maternal dietary patterns during pregnancy and the risk of acute leukemia in infants. The "Balanced & Vegetable-Rich" pattern, characterized by a balanced consumption of various food groups and higher vegetable intake, exhibited a negative association with acute leukemia when compared to the "High Dairy & Cereals" Pattern (adjusted odds ratio [OR] = 0.51; 95% confidence interval [CI]: 0.29, 0.90). We observed that mothers who gave birth to girls and adhered to a healthy dietary pattern during pregnancy exhibited significantly lower odds of their children developing AL compared to those who gave birth to boys [OR = 0.32 (95% CI 0.11, 0.97)]. Our results underscore the significance of maternal nutrition as a modifiable factor in disease prevention and the importance of prenatal health education.

P14 Blueberry muffin syndrome secondary to acute myeloid leukaemia: a learning point

Abstract A baby was initially referred at 6 weeks developing multiple erythematous, bruise-like and light-brown subdermal lumps over the head, face, arms, legs and buttocks. She had been born following breech presentation by elective C-section, which had been complicated by trauma and blood loss. No other clinical concerns were raised at this time. Nonaccidental injury was considered; however, subcutaneous fat necrosis of the newborn was the diagnosis made by paediatric team following slightly raised calcium and suggestive ultrasounds features. Newer lesions continued to arise in the same areas over 6 weeks, now with difficulty in feeding and irritability. One small cervical lymph node could be palpated. This was atypical for subcutaneous fat necrosis of the newborn and an urgent dermatological option was sought. On review, the lesions were clinically in keeping with an infiltrative process (blueberry muffin syndrome) and an urgent skin biopsy, blood film and TORCH screen were requested. A 4-mm punch biopsy confirmed leukaemia cutis with evidence of myeloid differentiation. MLL (KMT2A) rearrangement was detected, which was also subsequently found on a bone marrow sample. A peripheral blood film identified a population for NG2-positive myeloid blasts (6%), raising the possibility of infant acute myeloid leukaemia (AML). A TORCH screen did not highlight any congenital infections. The baby was promptly commenced on chemotherapy for AML. This case highlights how presentation can be atypical, the importance of reviewing possible diagnoses and considering an early biopsy to enable prompt treatment if required.

Hematopoietic Stem Cell (HSC)-Independent Progenitors Are Susceptible to Mll-Af9-Induced Leukemic Transformation.

Infant acute myeloid leukemia (AML) is a heterogeneous disease, genetically distinct from its adult counterpart. Chromosomal translocations involving the KMT2A gene (MLL) are especially common in affected infants of less than 1 year of age, and are associated with a dismal prognosis. While these rearrangements are likely to arise in utero, the cell of origin has not been conclusively identified. This knowledge could lead to a better understanding of the biology of the disease and support the identification of new therapeutic vulnerabilities. Over the last few years, important progress in understanding the dynamics of fetal hematopoiesis has been made. Several reports have highlighted how hematopoietic stem cells (HSC) provide little contribution to fetal hematopoiesis, which is instead largely sustained by HSC-independent progenitors. Here, we used conditional Cre-Lox transgenic mouse models to engineer the Mll-Af9 translocation in defined subsets of embryonic hematopoietic progenitors. We show that embryonic hematopoiesis is generally permissive for Mll-Af9-induced leukemic transformation. Surprisingly, the selective introduction of Mll-Af9 in HSC-independent progenitors generated a transplantable myeloid leukemia, whereas it did not when introduced in embryonic HSC-derived cells. Ex vivo engineering of the Mll-Af9 rearrangement in HSC-independent progenitors using a CRISPR/Cas9-based approach resulted in the activation of an aberrant myeloid-biased self-renewal program. Overall, our results demonstrate that HSC-independent hematopoietic progenitors represent a permissive environment for Mll-Af9-induced leukemic transformation, and can likely act as cells of origin of infant AML.

Abstract 3943: Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML

Abstract Rearrangement of the mixed lineage leukemia (MLL) gene occurs in up to 10% of acute leukemias and is particularly prevalent in infant acute leukemias (~80% of infant ALL cases). MLL-rearranged (MLLr) leukemia is one of the high-risk types of leukemia with aggressive nature, resistance to therapy, and high frequency of early relapse, and currently there are no targeted therapies for its treatment. So, there is huge unmet medical needs for MLLr leukemias. The protein protein interaction (PPI) between menin and MLLr is critical to the pathogenesis of MLLr-driven leukemias by deregulation of the HOXA and MEIS1 genes. It has been demonstrated by both preclinical and clinical studies that blockage of this PPI with menin inhibitors has potential therapeutic effects on MLLr associated acute leukemias. NPM1 mutation (NMP1c) is a frequently mutated gene found in AML, and NMP1 mutated AML depends on menin-MLL interaction for leukemogenesis. Given the fact that wild type MLL and MLLr have similar leukemogenesis-driven target genes like HOXA and MEIS1, as well as the same binding pocket on menin, it was therefore conceived that menin inhibitor could be useful to the treatment of NPM-mutated AML, which was again supported by both preclinical and clinical data. Herein, we would like to report the discovery of our proprietary menin inhibitor. Taking advantage of a couple of key interactions with menin protein, our compounds has relatively low molecular weight for a challenging PPI target, however, demonstrated comparable to or even better potency than those menin inhibitors in clinical trials. Besides in vitro pharmacology data, the DMPK, PK/PD and in vivo efficacy in animal model will be discussed too. Citation Format: Taishan Hu, Zhilin Deng, Honghai Li, Xiaochu Ma, Quanrong Shen, Lei Zhang, Peihua Sun, Ye Hua, Byran Huang. Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML [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 3943.

Clinical and molecular-biologic features of infant acute leukemia

Acute leukemia (AL) in children up 1 year old is very rare disease, comprise less 5 % of all pediatric AL cases. The clinical course of infant AL is extremely aggressive with frequent extramedullar dissemination, initial hyperleukocytosis and MLL aberrations. Predominance of unfavorable clinical and laboratory features leads to unsatisfactory treatment results by modern curative protocols in children up to 1 year old: 4-year event-free survival for acute lymphoblastic leukemia is 50.9 ± 4.5 % and 3-year event-free survival for acute myeloid leukemia – 72–74 %. Optimization of therapeutic programs for infant AL with consideration to anatomy and physiologic features of infants, inclusion of targeted drugs (blinatumomab), epigenetic and cell technologies is actual and not resolved problem in modern pediatric oncology-hematology.

3114 – FROM MECHANISMS OF LEUKAEMOGENIC DYSREGULATION TO NEW CANDIDATE THERAPEUTICS

Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities. Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities.

A B-cell developmental gene regulatory network is activated in infant AML.

Infant Acute Myeloid Leukemia (AML) is a poorly-addressed, heterogeneous malignancy distinguished by surprisingly few mutations per patient but accompanied by myriad age-specific translocations. These characteristics make treatment of infant AML challenging. While infant AML is a relatively rare disease, it has enormous impact on families, and in terms of life-years-lost and life limiting morbidities. To better understand the mechanisms that drive infant AML, we performed integrative analyses of genome-wide mRNA, miRNA, and DNA-methylation data in diagnosis-stage patient samples. Here, we report the activation of an onco-fetal B-cell developmental gene regulatory network in infant AML. AML in infants is genomically distinct from AML in older children/adults in that it has more structural genomic aberrations and fewer mutations. Differential expression analysis of ~1500 pediatric AML samples revealed a large number of infant-specific genes, many of which are associated with B cell development and function. 18 of these genes form a well-studied B-cell gene regulatory network that includes the epigenetic regulators BRD4 and POU2AF1, and their onco-fetal targets LIN28B and IGF2BP3. All four genes are hypo-methylated in infant AML. Moreover, micro-RNA Let7a-2 is expressed in a mutually exclusive manner with its target and regulator LIN28B. These findings suggest infant AML may respond to bromodomain inhibitors and immune therapies targeting CD19, CD20, CD22, and CD79A.

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.

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.

Genetic and Epigenetic Characterization of a Discordant KMT2A/AFF1-Rearranged Infant Monozygotic Twin Pair.

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment–genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

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.

Infant Acute Myeloid Leukemia with t(X;6)(p11;q23)/MYB-GATA1- a First Female Case in the Entity

Introduction T(X;6)(p11;q23) is a rear but recurrent chromosomal translocation in infant acute myeloid leukemia (AML). It leads to the formation ofMYB-GATA1fusion gene that interferes with granulocyte lineage commitment thus causing AML preliminary with basophilic phenotype. Only males were seen previously in this entity [Dastugue et al. 1997, Belloni et al. 2011, Quelen et al. 2011]. Here we report extensive molecular characterization of 2 infant AML cases with t(X;6)(p11;q23)/MYB-GATA1enrolled in the Russian AML registration study with one of them being a girl. Case reports A boy aged 5,5 mo. with WBC 27,7*109/L and 45% blasts in bone marrow was diagnosed AML, M5a by morphological, cytochemical and immunological observations. He was treated according to AML-BFM-2004 protocol and received allogenic HSCT from matched unrelated donor in the first remission. Patient stays in complete remission for 4 years now. A girl aged 8 mo. was headed to a hospital with febrile fever and petechiae. She had initial WBC 33,8*109/L and 35% bone marrow blasts of basophilic phenotype. Spinal tap confirmed CNS involvement (cytosis 2,6/mm3, 3% blasts). Patient received induction therapy (cytarabine, etoposide, daunorubicin) and achieved a complete remission. However, she developed severe myelodepression and died of infection 3 mo. after the diagnosis. Results MYB-GATA1.GTG-banded karyotyping showed a rearrangement between chromosomes 6 and X as a sole abnormality in both patients - insertion of chromosome 6 material into Xp11 band in a boy and reciprocal translocation t(X;6)(p11;q23) in a girl. FISH for relevant age-specific translocations, includingKMT2Arearrangements and t(7;12)(q36;p12)/ETV6-HLXB, was negative. RNA-seq (TruSeqRNA, Illumina, San-Diego, CA, USA) revealedMYB-GATA1fusion expression with exon 8 - exon 5 junction identical in both patients which was validated by RT-PCR and Sanger sequencing. Direct genomic PCR confirmed breakpoints withinMYBintron 8 andGATA1intron 4 and uncovered patient-specific junctions (see Figure). Reciprocal fusions.The formation of reciprocal fusions was also shown in two studied patients. A boy demonstratedGATA1-TUBE(6q21) intron 4 - intron 7 junction. It is non-functional due to tail-to-tail transcriptional orientation. Such structures are likely to form sinceMYBandGATA1are located in an opposite genomic orientation, so our finding goes in line with previous unsuccessful attempts to look for reciprocalGATA1fusion transcripts expression [Quelen et al. 2011]. However, a girl patient had reciprocalGATA1-CD164fusion (exon 4 - exon 6) actively transcribed together with third fusionMYB-CD164. On the one hand, this confirms that additional event such as inversion of either chromosomal fragment is required for t(X;6)(p11;q23) to manifest. On the other hand, this is, to our knowledge, the first demonstration of reciprocalGATA1fusion transcript expression inMYB-GATA1-associated AML. Wild-type MYB and GATA1 expression.AlongsideMYB-GATA1expression both patients demonstrated the overexpression of wild-typeMYBby qPCR. This is in agreement with MYB overexpression being a feature of undifferentiated cells and diminishing during differentiation [Bartunek et al. 2003]. Wild-typeGATA1expression (its lossper se) is of key importance inMYB-GATA1-associated AL. It was previously proposed that the loss of wild-typeGATA1is essential for leukemogenesis in male patients as the only copy of this gene is disturbed by chromosomal translocation [Quelen et al. 2011]. We observed no wild-typeGATA1expression in our male patient. However, in the female patient wild-type GATA1 expression was present. Additional events.The samples were also screened for additional genetic events associated with myeloid neoplasia by targeted DNA NGS (QIAact Myeloid DNA UMI Panel, Qiagen, Hilden, Germany), fragment analysis (NPM1,FLT3-ITD) and Sanger sequencing (cKIT,FLT3-TKD). NoFLT3,NPM1and cKIT pathogenic variants were found. NoGATA1truncating mutations were found. Deleterious variants were found inNRASgene (p.G12C in a boy, p.G13D in a girl). Female patient also demonstrated pathogenic variants inKRAS(p.A59E) andJAK2(p.N683G). Conclusions Thus, here we report a first case of female patient with t(X;6)(p11;q23)/MYB-GATA1. This raises a question whetherGATA1insufficiency is the primary mechanisms of leukemogenesis in this genetic subgroup. Figure Disclosures No relevant conflicts of interest to declare.

Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPβ deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPβ expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPβ, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

The results of allogeneic hematopoietic stem cell transplantation from a matched unrelated and haploidentical donors in children with high-risk infant leukemia in first and second remissions

Aсute myeloid leukemia (AML) in children aged 0–2 years and aсute lymphoid leukemia (ALL) up to 1 year (i.e., infants) frequently characterize high risk and poor prognosis. Аllogeneic hemopoietic stem cell transplantation (аllo-HCST) is a main curative but toxic option for these patients, and choice of allogeneic donor may be one of the important factor for long-term survival. Aim. To evaluate overall survival (OS), relapse free survival (RFS), transplant related mortality (TRM), "graft versus host" disease free/relapse free survival (GRFS) in infant with acute leukemia underwent allo-HCST from MUD vs haplodonor at 1st or 2nd remission. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University. 34 children with infant acute leukemia: 23 pts with AML (68%) and 11 – with ALL (32%) – underwent allo-HSCT from MUD vs haplo at 1st or 2nd remission between 2004–2018 were analyzed. Median age at allo-HCST – 22 months (6 months – 5 y.o.). HSCT was performed from MUD in 19 (56%) pts (group 1), haplo – 15 (44%) pts (group 2). Myeloablative conditioning received 29 (85%) pts. Reduced intensity conditioning received 5 (15%) pts. Posttransplant cyclophosphomyde (PtCy) was used in 10 (53%) pts in the group 1 and 14 (93%) pts. in the group 2 (p = 0.043). Engraftment was identified in 18 pts (95%) of group 1 and 12 pts (80%) of group 2 (p = 0.28). At the median follow up 3.5 years OS is 79% in the group 1 аnd 73% in the group 2 (p = 0.68). RFS is 79% in the group 1 аnd 67% in the group 2 (p = 0.41). GRFS is 39% in the group 1 аnd 47% in the group 2 (p = 0.5). TRM occurred in 2 pts (11%) of group 1 (due to infectious and toxicity) and no one of the group 2 (p = 0.2). Haplo-HSCT with PtCy is a good alternative to MUD with high efficacy and acceptable toxicity in children with infant acute leukemia at 1st or 2nd remission.

A Distinct Oncofetal B-Cell Transcriptional Program Is Activated in Infant Acute Myeloid Leukemia and Reveals Novel Therapeutic Strategies

Infants with acute leukemia present special challenges, due in part to the unusual features of their disease. A clonal oncofusion protein is detectable in the vast majority of infants presenting with acute leukemia, whether myeloid (AML), lymphoblastic (ALL), or mixed-phenotype (MPAL). Many recurrent oncofusion proteins seen in this age group involve the KMT2A (formerly MLL, Mixed Lineage Leukemia) gene, located on chromosomal band 11q23, which is notorious for both lineage switching and poor clinical prognosis. Other fusions involve components of the nuclear pore complex (NUP98), chromatin modifiers (KAT6A), or lineage-associated pioneer factors (such as MYB and GLIS2). This diversity of oncofusion proteins has hampered efforts to develop targeted agents, and over half of infant cases eventually succumb to their disease. Previous work showed that infant ALL is distinct from childhood or adult ALL. Here we show that infant AML is similarly distinct from AML in other age groups, and this difference yields transformative opportunities to repurpose existing targeted and immune therapies. mRNA sequencing (RNAseq) analysis of over 1500 participants in Children's Oncology Group clinical trials revealed hundreds of infant-AML-specific transcripts, many associated with early B cell development. 18 of the affected genes form a canonical B-cell regulatory circuit, including the epigenetic regulators BRD4 and POU2AF1, and their onco-fetal targets LIN28B and IGF2BP3. These four genes also host hypo-methylated super-enhancers in most infant AML cases, but in few older patients. The MYC proto-oncogene is directly implicated in a regulatory loop involving microRNA let7a-2, which is expressed in a mutually exclusive manner with its target and regulator LIN28B (Fig 1A). By contrast, the WT1 gene, abundantly expressed in over 90% of AML cases from other age groups, is transcribed at low levels in most infant AML cases. Single-sample gene set enrichment analysis revealed that most cases of infant AML bear more similarity to infant B-cell ALL or MPAL than to AML in older children, adolescents, and adults (Fig 1B). Higher-order chromatin conformation further emphasizes these differences. The MYC proto-oncogene, a critical node in the gene regulatory network (Fig. 1A) observed across infant acute leukemia cases, is routinely amplified via trisomy 8 in this age group (and in fact all childhood leukemia). The IGF2BP3 gene, another critical node (Fig. 1A), is rarely deleted in infants (deletions of 7q tend to spare 7p15, and complex karyotype cases often retain two or more copies on derivative chromosomes). The immature humoral and adaptive immune system in infants complicates treatment in this age group, but also yields metabolic vulnerabilities which can be exploited to break the nearly ubiquitous positive feedback loop described herein. These age-specific differences suggest that alternative therapeutic targets and modalities are warranted not only in infant AML, but across infant acute leukemia more generally. The relatively small numbers of infant acute leukemia cases by lineage, along with common mechanisms that appear to sustain most cases of infant acute leukemia, and frequent clinical reports of lineage switching in infants regardless of morphology at presentation, all support this contention. Our results indicate that combination therapies coupling experimental ages (such as DFMO, which targets metabolic regulation of MYC, LIN28B, and let-7a via ornithine decarboxylase, and has shown substantial efficacy in MYCN-amplified neuroblastoma) with already approved agents such as BET inhibitors (which target BRD4) and blinatumomab (targeting CD19 as a canonical B-cell antigen) may be warranted. These immune-targeting combinations represent promising and potentially lineage-agnostic approach to improving outcomes for infants with acute leukemia, a group of patients for whom novel therapeutic strategies are desperately needed, and for whom less toxic, more effective, targeted combination therapies may spare a lifetime of late effects. Disclosures Pardo: Hematologics, Inc: Employment. Kaeding:Celgene: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership. Farrar:Novartis: Research Funding.

Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.