Megakaryoblastic Leukemia Research Articles

Abstract 3393: A novel targetable G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion in acute megakaryoblastic leukemia (AMKL)

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease, and targeted therapy has been shown to improve the outcomes of AML patients. Recently, we identified a novel G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion gene by RNA sequencing in a patient with acute megakaryoblastic leukemia (AMKL). G3BP1-CSF1R (GC) juxtaposed exon 8 of G3BP1 to exon 12 of CSF1R and was predicted to encode a fusion protein containing the disrupted juxtamembrane domain (JMD) and the intact kinase domain of CSF1R. A similar CSF1R rearrangement has been reported in an AMKL cell line, MKPL1, which harbors the RBM6-CSF1R fusion. As the second CSF1R fusion discovered in AMKL, it remains unclear whether GC is a novel oncogenic driver and whether it is involved in dictating megakaryocytic phenotype. The aim of this study was to characterize the GC fusion and to investigate its role in AMKL pathogenesis and its targetability. Immunofluorescence studies showed that GC and G3BP1 were located in the cytoplasm, unlike the wide-type CSF1R which exhibited vesicular localization. Co-IP studies indicated that GC could exist as dimers in the absence of ligand stimulation. Two cytokine-dependent cell lines (TF1 and M07E) transduced with GC lentivirus showed cytokine-independent growth and constitutive activation of CSF1R and its downstream signaling, indicating the transforming ability of GC. Consistently, NSG mice transplanted with GC-expressing TF1 cells developed AML. In vitro drug studies revealed that GC-transformed TF1 (TF1-GC) and MKPL1 cells were sensitive to dasatinib and pexidartinib, as demonstrated by inhibition of kinase activation, reduced cell proliferation, and increased apoptosis. Moreover, in NSG mice transplanted with TF1-GC cells, dasatinib and pexidartinib significantly decreased leukemia burden and prolonged survival. Using flow cytometry, we observed that GC increased cell size, granularity, and megakaryocytic markers (CD41 and CD61) in M07E, TF1, and human cord blood CD34+ stem and progenitor cells, suggesting that GC could promote megakaryocytic skewing. Taken together, we identified and characterized a novel targetable GC fusion in AMKL. These findings may provide insights into the role of CSF1R in AMKL pathogenesis and a rationale for CSF1R-targeted therapy in AML patients with CSF1R activating alterations. Citation Format: Xi Luo, Chi Keung Cheng, Hoi Yun Chan, Yuet Fong Kam, Kam Tong Leung, Chi Kong Li, Margaret Heung-Ling Ng. A novel targetable G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion in acute megakaryoblastic leukemia (AMKL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3393.

SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons.

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells

To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

Features of acute megakaryoblastic leukemia diagnosis in a child with Down syndrome

We present a description of the difficulties in diagnosing acute megakaryoblastic leukemia in a child with Down syndrome aggravated by multiple comorbidities. In this case, a comprehensive assessment of clinical data, the results of an automatic complete blood count with a detailed interpretation of the entire range of parameters, as well as morphological and immunophenotypic bone marrow examination using an extended panel of monoclonal antibodies played a key role in the diagnosis.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

Mixed-phenotype acute leukemia, T/megakaryoblastic: does it really exist?

Mixed-phenotype acute leukemias (MPAL) account for < 4% of all cases of acute leukemias. These are a heterogeneous group of leukemias grouped together by the WHO classification as "rare subtypes." The diagnosis and treatment of MPAL is extremely challenging particularly for low middle income countries. Of these, B/myeloid and T/myeloid combinations are relatively common subtypes. However, megakaryoblastic and erythroid lineages in combination with other lineages are still rare enough to not even be addressed in the WHO classification. To date, there have been only a few reports of mixed B or T cell and megakaryocytic or mixed B or T cell and erythroid leukemias. We report the clinical presentation, diagnostic profile, and disease course of MPAL cases with a biphenotypic pattern consistent with T/megakaryoblastic lineage which is not yet defined in WHO classification. These cases were phenotyped using 8-color flow cytometry (BD FACS CANTO-II) using an extensive panel of markers. Interphase fluorescence in situ hybridization (FISH) was done using dual color dual fusion probes for BCR::ABL1, RUNX1::RUNX1T1, and ETV6::RUNX1, while MLL and CBFB gene rearrangement was tested by break-apart probes. Karyotyping was performed using the conventional GTG-banding technique. Both FISH and karyotyping were analyzed by the automated cell imaging system Leica Biosystems, using Cytovision MB8. The cases presented here satisfy the criteria for both T-lineage assignment (cyCD3 intensity reaches that of normal T-lymphocytes) and acute megakaryoblastic leukemia (≥ 1 megakaryocytic marker in > 50% blasts) and thus represent the first documented examples of this unusual entity from Pakistan. It is crucial to report these cases to gather more data about clinical presentation, diagnostic profile, and disease course. Additionally, the reported cases highlight the limitations of existing classifications which do not address rare subtypes. More importantly, T/megakaryoblastic MPAL needs to be included in the WHO classification as a separate entity.

Haematopoietic cell transplantation for children with acute megakaryoblastic leukaemia without Down syndrome.

Patients with acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL) have an excellent survival rate; however, patients with non-DS-AMKL experience poor outcomes. Therefore, this study retrospectively analysed 203 children with non-DS-AMKL who underwent their first haematopoietic cell transplantation (HCT) from 1986 to 2015 using a nationwide Japanese HCT registry data to assess HCT outcomes for non-DS-AMKL. The 5-year overall survival (OS) and event-free survival (EFS) rates were 43% and 38% respectively. The 5-year OS rate was significantly higher for patients who underwent HCT in the first complete remission (CR1, 72%) than for those in the second CR (CR2, 23%) and non-CR (16%) (p < 0.001), and for those from a human leukocyte antigen (HLA)-matched (52%) than for those from an HLA-mismatched donor (27%) (p < 0.001). Multivariate analysis for OS revealed that HCT in CR2 and non-CR was a significant risk factor (hazard ratio, 5.86; 95% confidence interval, 3.56-9.53; p < 0.001). The 3-year EFS in patients who received HCT in CR1 using reduced-intensity conditioning (RIC, 35%) was significantly lower than in those using myeloablative conditioning (busulfan-based, 71%; total body irradiation-based, 58%) (p < 0.001). Risk stratification in patients with non-DS-AMKL should be established to determine HCT indication in CR1.

The diagnostic value of cytogenetics in a patient with concurrent mediastinal non-seminomatous germ cell tumour (NSGCT), fibrolamellar hepatocellular carcinoma and acute megakaryoblastic leukaemia (AMKL) associated with widespread bone marrow necrosis

Background: The rare association between NSGCT and AMKL has been reported since 1985.1,2 Isochromosome 12p is the most common cytogenetic abnormality in NSGCT but is only rarely found in AMKL unless in association with NSGCT.3

Acute promyelocytic leukemia with FIP1L1::RARA fusion gene: The clinical utility of transcriptome sequencing and bioinformatic analyses.

Acute promyelocytic leukemia (APL) is typically characterized by the presence of coagulopathy and the PML::RARA fusion gene. The FIP1L1::RARA has been reported as a novel fusion gene, but studies on its pathogenesis are limited. A FIP1L1::RARA fusion in a child finally diagnosed as APL was reported. RNA sequencing (RNA-seq) of six patients (three cases of acute lymphoblastic leukemia (ALL), one case of myelodysplastic syndrome (MDS), one case of acute megakaryoblastic leukemia (M7), and one case of APL with FIP1L1::RARA) were performed. Transcriptome analysis of six patients was performed by RNA-seq. The heat map was used for showing the RNA expression profile, the volcano plot for identifying differential expression genes (DEGs), and the KEGG Orthology-Based Annotation System (KOBAS) online biological information database for KEGG pathway enrichment analysis. Obvious differences between APL with FIP1L1::RARA and hematologic malignancies were identified. 1060 common differentially expressed genes (co-DEGs) were detected between APL with FIP1L1::RARA vs ALL and APL with FIP1L1::RARA vs myeloid neoplasms (MDS, M7), the up-regulated genes were mainly mapped into platelet activation, cancer, AMPK signaling pathway, PI3K-Akt signaling pathway, and MAPK signaling pathway. The down-regulated genes were significantly associated with TNF signaling pathway, Rap1 signaling pathway, Age-RAGE signaling pathway, and apoptosis. A FIP1L1::RARA fusion in a child finally diagnosed as APL was reported. RNA-seq may provide a new diagnostic method when RARA rearrangements fail to be identified by conventional methods. In the analysis of co-DEGs between case vs ALL and case vs myeloid neoplasms, the up-regulated and down-regulated genes were enriched in different signaling pathways. Further experimental studies are needed to identify pathogenesis and treatment for APL with FIP1L1::RARA.

Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia

AbstractMyeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Lineage switch in a pediatric patient with KMT2A-MLLT3 from acute megakaryoblastic leukemia to T cell acute lymphoblastic leukemia at the fourth relapse after allo-HSCT: with literature review.

We present a patient with acute megakaryoblastic leukemia (AMKL) harboring KMT2A-MLLT3 that converted to T cell acute lymphoblastic leukemia (T-ALL) at her fourth relapse. A 4-year-old girl developed AMKL with multiple swollen lymph nodes. She exhibited several recurrences in the bone marrow and died of septic shock after her fourth relapse. Bone marrow cells at the initial diagnosis and at all four relapses had the same KMT2A-MLLT3 fusion transcript. She also developed a somatic mutation (c.7177C > T p.Q2393X) of NOTCH1 at the fourth relapse. This sequential phenotypic and cytogenetic study may yield valuable insights into the mechanism of AMKL to T-ALL lineage switch and possible implications for treatment selection.

Erythroid/Megakaryocytic Differentiation Confers BCL-XL Dependency and Venetoclax Resistance in Acute Myeloid Leukemia

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia (PEL), myelodysplastic syndrome (MDS) with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Hypothesizing that the distinct lineage of acute myeloid leukemia (AML) with erythroid or megakaryocytic differentiation may confer specific vulnerabilities, we sought to identify selective dependencies in these rare subtypes in comparison with other AML types. We show that AML cells with erythroid or megakaryocytic differentiation depend on the anti-apoptotic protein BCL-XL, rather than BCL-2, using a combination of ex vivo drug sensitivity and resistance testing, genetic perturbation, transcriptomic profiling and mouse xenograft experiments. To identify druggable dependencies in erythroid and megakaryoblastic leukemias, we screened over 500 drugs across a 10,000-fold concentration range in four erythroid and two megakaryoblastic AML cell lines and used other AML cell lines as controls (Figure A). The high-throughput screens identified BCL-XL-selective inhibitors including A-1331852 as highly effective against erythroid and megakaryoblastic leukemia cell lines in comparison with 15 cell lines representing other AML types (Figure B). In contrast, the erythroid/megakaryoblastic AML subtypes were resistant to the BCL-2 inhibitor venetoclax used clinically in the treatment of AML (Figure A). To confirm the on-target mechanism of BCL-XL inhibition, we next evaluated the genetic dependencies of erythroid and megakaryoblastic leukemias using public genome-scale CRISPR-Cas9 and RNAi screening data from 5 erythroid, 3 megakaryoblastic, and 15 other AML cell lines. This analysis demonstrated striking essentiality of BCL2L1 encoding BCL-XL, but not BCL2 or MCL1, for the survival of erythroid and megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared to other subtypes of AML and other hematological malignancies, providing a basis for the observed functional dependencies. BCL-XL inhibition effectively killed bone marrow cells of 5 out of 8 AML patient samples with erythroid or megakaryocytic differentiation ex vivo, with IC50 ranging from 0.6 to 55 nM. In contrast, only one out of 40 other AML patient samples was sensitive (IC50 < 60 nM) to BCL-XL inhibition. To better understand the drug sensitivity and gene expression profiles of the blasts, we performed integrated single-cell transcriptomics and ex vivo flow cytometry-based drug sensitivity profiling on mononuclear cells from two patients with erythroid/megakaryocytic AML. The blasts showed enriched BCL2L1 expression compared to other cell populations, providing a basis for the increased sensitivity of the blasts to BCL-XL inhibition and venetoclax resistance observed also at the single-cell resolution. In a mouse xenograft model using the HEL erythroleukemia cell line, treatment with the BCL-XL inhibitor A-1331852 effectively reduced tumor burden. To find treatment regimens enabling long-term disease control, we explored potential combination treatments by testing 6 other compounds with the BCL-XL inhibitor A-1331852 in 8x8 concentration matrices. Combining BCL-XL inhibition with the JAK inhibitor ruxolitinib, the BCL-2 inhibitor venetoclax, or the hypomethylating agent azacitidine showed potential for more durable responses both in a 3-day ex vivo assay as well as long-term treatments spanning over a month. Collectively, our results suggest targeting BCL-XL as a potential therapy option in erythroid and megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Vivo Crispr Screening Identifies Novel Vulnerabilities in ETO2/GLIS2-rearranged Leukemia

Background: CRISPR screening has allowed for massively parallel interrogation of the genome for vulnerabilities in a variety of cancer models. However, these experiments are largely performed in vitro due to the scale and conditions required to effectively conduct these screens. Furthermore, there still remain genetic subtypes of acute leukemia which are poorly represented among existing cell lines. We sought to determine the feasibility of conducting screens with focused libraries in patient-derived leukemia xenografts. Methods: We utilized a patient-derived acute megakaryoblastic leukemia (AMKL) bearing an ETO2/GLIS2 fusion, representing an aggressive subtype of pediatric acute leukemia with limited comparable cell lines (Gruber TA, et al. Cancer Cell 2012.) Based on its transplantability in NOD.scid.Il2Rγcnull-SGM3 (NSGS) mice vs culture in vitro, we performed all subsequent experiments in vivo. We first sought to establish stable Cas9 expression, which we successfully achieved by lentiviral transduction followed by FACS-enriched transplant, followed by a second round of FACS enrichment with limiting dilution transplant. As judged by mCherry flow cytometry, we achieved highly pure (>90%) Cas9-expressing populations, which we verified by western blotting. We further verified functionality of these cells by verifying cutting at the safe harbor locus AAVS1. In order to identify molecular vulnerabilities in this system, we identified the top 100 differentially expressed genes (DEG) between ETO2/GLIS2-rearranged leukemias versus other leukemia PDXs which we previously characterized. We included eight transcription factors with motifs enriched in our DEG analysis, as well as eight pan-essential positive controls (such as MYC, BRD4). Four guide RNAs (gRNA) per gene were selected from the Broad Brunello genome-wide library (Doench JG, et al. Nat Biotechnol 2016) as well as 50 "safe targeting" control guides (Morgens DW, et al. Nat Commun 2017) directed against non-essential genomic regions for a total of 518 guides. Guides were ordered as pooled oligonucleotides, cloned into a lentiviral gRNA backbone using standard techniques, then used to prepare lentivirus via transient transfection of 293T cells followed by titration to achieve an MOI of 0.3. With this MOI and an approximate engraftment frequency of 2% following intrafemoral injection, we transplanted 8.7e7 cells across eight NSGS mice to achieve adequate starting representation of our pooled library. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by Illumina deep sequencing, and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). Results: One mouse per cohort was sacrificed at 24 hours post-transplant and sequenced for gRNA representation, which confirmed that library representation was largely preserved with our approach. At the time of sacrifice, spleen and bone marrow samples were independently sequenced, but no major bias in guide dropouts was observed between these compartments. Subsequent analyses were performed on the marrow alone. We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs as well as targets previously described to be essential through our own orthogonal experiments (BCL2) or previously published findings (GLIS2, GATA3) (Thirant C et al. Cancer Cell 2017). Among transcription factors found to be essential, we identified several which had not previously been strongly associated with acute leukemia (FOXD4L1, ZNF740). Conclusion: Patient-derived xenografts represent a unique platform for targeted functional genomic interrogation of acute leukemias. These screens can be performed with a tractable number of animals and modest sequencing requirements. Additional experiments are ongoing to identify the mechanistic implications of the vulnerabilities described here in ETO2/GLIS2-rearranged AMKL.

High Fatality CBFA2T3-GLIS2 Pediatric Acute Megakaryoblastic Leukemia Is Sensitive to BCL-XL Inhibition

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of pediatric acute myeloid leukemia (AML) with dismal survival prognosis in children below 3 years of age (<40% of cure rates). In two thirds of cases, recurrent and mutually exclusive oncogenic fusions are detected, such as CBFA2T3-GLIS2 (CG2) or NUP98 rearrangements (e.g. NUP98-KDM5A), which are considered the transformative event in this disease. The development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material, which is among other things attributed to myelofibrosis and the rarity of primary patients. Therefore, our research group has engineered synthetic human models of high-fatality pediatric AMKL with different oncogenic fusions that faithfully phenocopy the disease in a patho-physiological context. In recent years, inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, entered the therapeutic arena in adult AML and pediatric leukemia with promising results. The molecules emphasized on are Venetoclax (a specific inhibitor of BCL2) and Navitoclax (broader affinity to BCL2, BCL-XL and BCL-W). Even though we see promising results in other leukemia, these agents were so far not investigated as potential therapies in pediatric high-fatality AMKL. To first overcome the paucity of patient material for research, we generated synthetic models of high-risk AMKL by transducing cord-blood derived hematopoietic stem and progenitor cells (HSPC) with lentiviral particles carrying the CBFA2T3-GLIS2 oncogenic fusion, followed by xenotransplantation into NSG mice. This approach generated leukemia with a latency between 9.9 and 36.1 weeks in primary mice which are able to sustain serial rounds of transplantation in recipient mice (up to 5 rounds tested). Furthermore, leukemic blasts immunophenotypically recapitulate AMKL, demonstrating expression of megakaryopoietic markers such as CD41, CD61 and CD56 that are even maintained after in vitro culture of 6 days and subsequent transplantation. Furthermore, we derived a CG2 specific gene expression signature from transcriptomic profiling of our models as well as patient samples that showed high expression of NCAM1, BMP2, ERG and low GATA1 levels, as reported for CG2 leukemia. Remarkedly, one of the specific up regulated genes in our signature for CG2 was pro-survival factor BCL2. In addition, our models are located in the megakaryocytic-erythroid differentiation space, as assessed by single cell RNAseq, and similar to their healthy counterparts of the megakaryopoietic lineage they demonstrated high expression of BCL-XL on RNA and protein level, in contrast to AML samples. Based on the aberrant expression of BCL2 in our synthetic leukemia models and lineage related expression of BCL-XL, we set out to test Venetoclax and Navitoclax in the context of AMKL. We identified Navitoclax (inhibitor of BCL2, BCL-XL and BCL-W) as a therapeutic vulnerability in our models of AMKL (CG2 and NUP98 rearranged), whereas our cells demonstrated resistance to treatment with Venetoclax. We further investigated the molecular mechanism and showed that shRNA mediated knock-down or inhibition of BCL-XL, by either Navitoclax or the BCL-XL specific proteasomal degrader DT2216, results in significant induction of apoptosis in our models of AMKL. On the contrary, genetic or pharmacological inhibition of BCL2 did not induce apoptosis in our synthetic models of AMKL, even at doses as high as 10µM. Furthermore, a genotype matched leukemic model of NUP98-KDM5A, which was presenting as monocytic (CD68+LYZ+) AML instead of AMKL, was resistant to either treatment with Venetoclax or Navitoclax whereas ML2, a MLL rearranged AML cell line, showed increased sensitivity to both Venetoclax and Navitoclax treatment. Moreover, in vivo treatment of a xenografted CG2 AMKL model with Navitoclax reduced leukemic burden in mice in bone-marrow and spleen in comparison to vehicle treated controls. In conclusion, we generated a model of rare CG2 pediatric high-fatality leukemia that faithfully mimics the patient situation and allows biomass generation for large scale multi-omic approaches. Furthermore, we demonstrate a lineage- and genotype specific targetable dependency of pediatric AMKL towards inhibition of BCL-XL but not BCL2, that could be transferred to the clinic as a novel therapeutic option in high-risk infant leukemia.

Acute Panmyelosis with Myelofibrosis in an Elderly Patient- an Uncommon Cause of Pancytopenia

Introduction: Acute panmyelosis with myelofibrosis (APMF) is rare yet a fatal hematological malignancy. It had been classified as a subtype of acute myeloid leukemia (AML) under the category "AML, not otherwise specified" in the WHO classification of hematological tumors. APMF usually presents with cytopenia without any marked splenomegaly. We report a case of an 80-year-old female who presented with generalized weakness. An extensive workup unmasked the presence of acute panmyelosis with myelofibrosis. Case Report: An 80-year-old female patient with history of epilepsy, essential hypertension, and hyperlipidemia presented to the clinic for generalized weakness and confusion for two weeks. She was also having dyspnea, initially on exertion, which progressed to even at rest for the last one week and was unable to ambulate without assistance. The patient also reported easy bruising with minor trauma and bruises on the upper extremity. The patient denied having any fever, chills, chest pain, orthopnea, dizziness, loss of consciousness, history of bleeding, or weight loss. On physical examination, the patient did not have enlarged lymph nodes or hepatosplenomegaly. Initial labs showed hemoglobin of 4.8 g/dL, platelets 5K/uL, a white blood count of 1.8 K/uL with differentials of 49% segmented neutrophils, 0.85 K/uL absolute neutrophils, 4% band cells, 44% lymphocytes, 1% myelocytes, 1% nucleated RBC's, 0.09 K/uL absolute monocytes, and 3% monocytes. Her absolute reticulocyte count was 0. Red blood cells were hypochromic with anisopoikilocytosis. The coagulation profile showed PT 16.5 seconds, INR 1.4, and aPTT 37 seconds. She also underwent a CT head for the confusion which was unremarkable. Chest x-ray revealed left mid & lower zone infiltrates consistent with pneumonia and the patient was given ceftriaxone. The patient was transfused irradiated packed red blood cells. There was a concern for bone marrow disorders such as myelodysplastic syndrome and leukemia, so the patient underwent a bone marrow biopsy. Flow cytometry of bone marrow aspirate revealed no significant blast cells; however, it showed myeloid neoplasm with myelofibrosis and proliferation of the atypical megakaryocytes with variable hypercellularity and diffusely marked reticulin fibrosis. Immunohistochemistry was positive for E-cadherin on megaloblastic erythroid, myeloperoxidase (highlighted patchy foci of maturing myeloid cells), CD34 (widespread positive in the atypical megakaryocyte, but not blast cells), and CD61 (GPIIIA). These findings along with the absence of splenomegaly were consistent with Panmyelosis with myelofibrosis. After the family discussion about chemotherapy and bone marrow transplant, a combined decision was made for hospice with comfort measures and the patient died within a week. Discussion: Acute panmyelosis with myelofibrosis is not thoroughly studied. Although WHO has classified it as a type of AML, some still consider it equivalent to AMKL or an acute kind of MDS (myelodysplastic syndrome). APMF comprises less than 1% of the cases of AML. The disease typically presents with an acute onset of pancytopenia without any splenomegaly and is rapidly fatal with mean survival of less than a year. Significant differential diagnoses include acute megakaryoblastic leukemia (AML) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC). AML and APMF share multiple features like fibrotic un-aspirable bone marrow and the presence of megakaryoblasts. However, in AML, most of the blast population comprises megakaryoblasts, while in APMF, non-megakaryocytic cells are predominant. Compared to AML-MRC, abrupt clinical onset and worse survival rate point towards APMF. In addition, multilineage dysplasia led by myelodysplastic syndrome is also characteristic of AML-MRC. Similarly, exposure to leukemogenic therapeutic agents should also be ruled out, as such cases should be categorized as MDS or AML therapy-related diseases. Due to the disease being extremely rare, there are no established management protocols. The most commonly used treatment is with 3 + 7 induction chemotherapy. A retrospective study showed a poor outcome of hematopoietic cell transplantation due to a high relapse rate. Another case showed remission by 3+7 induction with idarubicin and cytarabine followed by cytosine arabinoside. In our case, hospice care was preferred considering the age and comorbidities

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

JAK: Not Just Another Kinase.

The JAK/STAT axis is implicated in cancer, inflammation, and immunity. Numerous cytokines/growth factors affect JAK/STAT signaling. JAKs (JAK1, JAK2, JAK3, and TYK2) noncovalently associate with cytokine receptors, mediate receptor tyrosine phosphorylation, and recruit ≥1 STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6). Tyrosine-phosphorylated STATs dimerize and are then transported into the nucleus to function as transcription factors. Signaling is attenuated by specific suppressor of cytokine signaling proteins, creating a negative feedback loop. Both germline mutations and polymorphisms of JAK family members correlate with specific diseases: Systemic lupus erythematosus (TYK2 polymorphisms); severe combined immunodeficiency (JAK3 mutations); pediatric acute lymphoblastic leukemia (TYK2 mutations); and hereditary thrombocytosis (JAK2 mutations). Somatic gain-of-function JAK mutations mainly occur in hematologic malignancies, with the activating JAK2 V617F being a myeloproliferative disorder hallmark; it is also seen in clonal hematopoiesis of indeterminate potential. Several T-cell malignancies, as well as B-cell acute lymphoblastic leukemia, and acute megakaryoblastic leukemia also harbor JAK family somatic alterations. On the other hand, JAK2 copy-number loss is associated with immune checkpoint inhibitor resistance. JAK inhibitors (jakinibs) have been deployed in many conditions with JAK activation; they are approved in myeloproliferative disorders, rheumatoid and psoriatic arthritis, atopic dermatitis, ulcerative colitis, graft-versus-host disease, alopecia areata, ankylosing spondylitis, and in patients hospitalized for COVID-19. Clinical trials are investigating jakinibs in multiple other autoimmune/inflammatory conditions. Furthermore, dermatologic and neurologic improvements have been observed in children with Aicardi-Goutieres syndrome (a genetic interferonopathy) treated with JAK inhibitors.

Pediatric non-Down's syndrome acute megakaryoblastic leukemia patients in China: A single center's real-world analysis.

Non-Down’s syndrome acute megakaryocytic leukemia (non-DS-AMKL) is a subtype of childhood acute myeloid leukemia (AML), whose prognosis, prognostic factors and treatment recommendations have not yet to be defined in children. We conducted a retrospective study with 65 newly diagnosed non-DS-AMKL children from August 2003 to June 2020 to investigate the clinical impact of factors and clinical outcome. Among all 65 patients, 47 of them were treated at our center who received three different regimens due to time point of admission (CAMS-another, CAMS-2009 and CAMS-2016 protocol), and the efficacy were compared. Patients with newly diagnosed non-DS-AMKL accounted for 7.4% of pediatric AML cases. The median age of the patients was 18 months at diagnosis, and over 90% of them were under three-years-old. The overall survival (OS) rates were 33.3% ± 1.7%, 66.7% ± 24.4% and 74.2% ± 4.0% for three groups (CAMS-another, CAMS-2009 and CAMS-2016 regimen), respectively. In CAMS-2016 group, the complete remission (CR) rate after induction was 67.7% (21/31), while the total CR rate after all phases of chemotherapy was 80.6% (25/31). The 2-year survival probability did not significantly improve in patients underwent HSCT when compared with non-HSCT group (75.0% ± 4.7% vs. 73.9% ± 4.6%, p=0.680). Those who had a “dry tap” during BM aspiration at admission had significantly worse OS than those without “dry tap” (33.3% ± 8.6% vs. 84.0% ± 3.6%, p=0.006). Moreover, the results also revealed that patients with CD34+ had significantly lower OS (50.0% ± 6.7% vs. 89.5% ± 3.5%, p=0.021), whereas patients with CD36+ had significantly higher OS than those who were negative (85.0% ± 4.0% vs. 54.5% ± 6.6%, p=0.048). In conclusion, intensive chemotherapy resulted in improved prognosis of non-DS-AMKL children and subclassification may base on “dry tap” and immunophenotypic. Although some progress has been made, outcomes of non-DS-AMKL children remain unsatisfactory, especially in HSCT group, when compared with other AML types.

CLINICAL AND MOLECULAR REPORT OF A RARE RAM POSITIVE NON-DOWN SYNDROME INFANT CBFA2T3-GLIS2 NEGATIVE, PRESENTING THE DRIVER P210-BCR-ABL1

Pediatric Acute Megakaryoblastic Leukemia (AMKL) comprises 4%‒15% of Acute Myeloid Leukemia (AML) cases. Down Syndrome (DS)-AMKL usually displays a favorable outcome, whereas the prognosis of non-DS-AMKL is generally poor. Here we present the clinical and genomic data of a rare non-DS-AMKL infant with p210-BCR-ABL1 fusion, co-occurring with a high-risk WT1 mutation. The baby was RAM positive and CBFA2T3-GLIS2 negative, showing refractoriness to treatment. A 1-year-old girl was referred to the Hospital with a history of fever and motor developmental delay for four months. On admission, she presented ecchymosis, swelling, and organomegaly (spleen and liver). The WBC count was 26.5 × 10 3 /μL, hemoglobin level was 7.6 g/dL, platelet count was 25 × 10 3 /μL, and LDH was 2.887. At diagnosis, she showed bilateral myeloid sarcoma. Morphological analysis of the Bone Marrow (BM) showed clustered cytoplasmic blebs. Flow cytometry analysis revealed 83% of blast cells and a profile compatible with AMKL and RAM phenotype. The patient was treated under the high-risk AML-BFM-2004. Imatinib was incorporated into the treatment after BCR-ABL1 detection. However, minimal residual disease remained positive throughout the treatment, making BM transplantation unfeasible. Disease refractoriness was persistent even with the administration of Dasatinib, FLAG-IDA and Venetoclax. The patient died one year after diagnosis due to sepsis during BM aplasia. We performed G-banding, FISH, and Multiplex RT-PCR experiments. The molecular response was assessed by absolute quantification via RT-qPCR assays. Sanger sequencing and NGS were performed on a targeted approach. The karyotype was 46,XX,t(9;22)(q21;q34). We observed polyploidy (3‒20N). FISH in polyploid Nuclei (8N) showed four BCR-ABL1 fusion signals. In diploid metaphases, we observed two BCR-ABL1 signals. Multiplex RT-PCR detected the p210- BCR - ABL1 . RT-qPCR monitoring detected persistent high levels of BCR-ABL1 transcripts associated with disease refractoriness. Sanger sequencing showed no GATA1 mutation. NGS revealed a missense WT1 pathogenic mutation, and CBFA2T3-GLIS2 was not present. RAM phenotype is characterized by overexpression of CD56 and under-expression of CD38, CD45, and HLA-DR. This phenotype is characteristic of children, usually associated with AML-M7 (38%) and intermediate-risk cytogenetics not associated with prognostic subgroups. RAM is related to a poor prognosis with high MRD and induction failure rates compared to non-RAM patients. This phenotype is associated with the CBFA2T3-GLIS2 fusion, which is related to extramedullary involvement, a particular blast morphology, and a worse outcome as compared to fusion-negative AML pediatric patients, including a high frequency of disease refractoriness and relapse. NGS studies did not show CBFA2T3-GLIS2 in our patient. Thus, our results indicate that RAM+ associated with the driver p210-BCR-ABL1, even with specific targeted therapy, may lead to treatment resistance regardless of CBFA2T3-GLIS2+. Here we highlight for the first time the presence of p210-BCR-ABL1 associated with RAM phenotype in the absence of CBFA2T3-GLIS2 and disease refractoriness in a non-DS-AMKL infant.