Tretinoin Enhances the Effects of Chemotherapy in Juvenile Myelomonocytic Leukemia Using an Ex Vivo Drug Sensitivity Assay.

PURPOSE: Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric malignancy with myelodysplastic and myeloproliferative features. Curative treatment is restricted to hematopoietic stem-cell transplantation. Fludarabine combined with cytarabine (FLA) and 5-azacitidine (AZA) monotherapy are commonly used pre-transplant therapies. Here, we present a drug screening strategy using a flow cytometry-based precision medicine platform to identify potential additional therapeutic vulnerabilities. METHODS: We screened 120 dual- and 10 triple-drug combinations (DCs) on peripheral blood (n = 21) or bone marrow (n = 6) samples from 27 children with JMML to identify DCs more effectively reducing leukemic cells than the DCs' components on their own. If fewer leukemic cells survived a DC ex vivo treatment compared with that DC's most effective component alone, the drug effect was referred to as cooperative. The difference between the two resistant fractions is the effect size. RESULTS: We identified 26 dual- and one triple-DC more effective than their components. The differentiation agent tretinoin (TRET; all-trans retinoic acid) reduced the resistant fraction of FLA in 19/21 (90%) samples (decrease from 15% [2%-61%] to 11% [2%-50%] with a mean effect size of 3.8% [0.5%-11%]), and of AZA in 19/25 (76%) samples (decrease from 69% [34%-100+%] to 47% [17%-83%] with a mean effect size of 16% [0.3%-40%]). Among the resistant fractions, the mean proportion of CD38+ cells increased from 7% (0.03%-25%; FLA) to 17% (0.3%-38%; FLA + TRET) or from 10% (0.2%-31%; AZA) to 51% (0.8%-88%; AZA + TRET). CONCLUSION: TRET enhanced the effects of FLA and AZA in ex vivo assays with primary JMML samples.

The Clinical Landscape of NRAS- mutated Juvenile Myelomonocytic Leukemia-like Myeloproliferation Includes Children With Costello Syndrome.

Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive pediatric disorder characterized by pathologic myeloproliferation because of alterations in RAS pathway genes. NRAS -mutated JMML encompasses a broad range of clinical severity. Herein we describe 4 unique cases of NRAS -mutated JMML and JMML-like myeloproliferation, 2 with somatic mutations and 2 with germline mutations. These cases illustrate the diverse clinical and hematologic presentation of this subtype of JMML, including a very unusual example presenting with Auer rods. Lastly, this is the first report of patients with phenotypic Costello syndrome presenting with JMML-like myeloproliferation, highlighting an important clinical phenomenon that has not been previously described.

Clinical parameter-based prediction of DNA methylation classification generates a prediction model of prognosis in patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare heterogeneous hematological malignancy of early childhood characterized by causative RAS pathway mutations. Classifying patients with JMML using global DNA methylation profiles is useful for risk stratification. We implemented machine learning algorithms (decision tree, support vector machine, and naïve Bayes) to produce a DNA methylation-based classification according to recent international consensus definitions using a well-characterized pooled cohort of patients with JMML (n = 128). DNA methylation was originally categorized into three subgroups: high methylation (HM), intermediate methylation (IM), and low methylation (LM), which is a trichotomized classification. We also dichotomized the subgroups as HM/IM and LM. The decision tree model showed high concordances with 450k-based methylation [82.3% (106/128) for the dichotomized and 83.6% (107/128) for the trichotomized subgroups, respectively]. With an independent cohort (n = 72), we confirmed that these models using both the dichotomized and trichotomized classifications were highly predictive of survival. Our study demonstrates that machine learning algorithms can generate clinical parameter-based models that predict the survival outcomes of patients with JMML and high accuracy. These models enabled us to rapidly and effectively identify candidates for augmented treatment following diagnosis.

JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1ß.

Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1ß, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1ß receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1ß signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.

Induced Pluripotent Stem Cells to Model Juvenile Myelomonocytic Leukemia: New Perspectives for Preclinical Research.

Juvenile myelomonocytic leukemia (JMML) is a malignant myeloproliferative disorder arising in infants and young children. The origin of this neoplasm is attributed to an early deregulation of the Ras signaling pathway in multipotent hematopoietic stem/progenitor cells. Since JMML is notoriously refractory to conventional cytostatic therapy, allogeneic hematopoietic stem cell transplantation remains the mainstay of curative therapy for most cases. However, alternative therapeutic approaches with small epigenetic molecules have recently entered the stage and show surprising efficacy at least in specific subsets of patients. Hence, the establishment of preclinical models to test novel agents is a priority. Induced pluripotent stem cells (IPSCs) offer an opportunity to imitate JMML ex vivo, after attempts to generate immortalized cell lines from primary JMML material have largely failed in the past. Several research groups have previously generated patient-derived JMML IPSCs and successfully differentiated these into myeloid cells with extensive phenotypic similarities to primary JMML cells. With infinite self-renewal and the capability to differentiate into multiple cell types, JMML IPSCs are a promising resource to advance the development of treatment modalities targeting specific vulnerabilities. This review discusses current reprogramming techniques for JMML stem/progenitor cells, related clinical applications, and the challenges involved.

Juvenile xanthogranuloma in Noonan syndrome.

Noonan syndrome (NS) is one of the common RASopathies. While the clinical phenotype in NS is variable, it is typically characterized by distinctive craniofacial features, cardiac defects, reduced growth, bleeding disorders, learning issues, and an increased risk of cancer. Several different genes cause NS, all of which are involved in the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. Juvenile xanthogranuloma (JXG) is an uncommon, proliferative, self-limited cutaneous disorder that affects young individuals and may be overlooked or misdiagnosed due to its transient nature. A RASopathy that is known to be associated with JXG is neurofibromatosis type 1 (NF1). JXG in NF1 has also been reported in association with a juvenile myelomonocytic leukemia (JMML). As RASopathies, both NS and NF1 have an increased incidence of JMML. We report a 10-month-old female with NS who has a PTPN11 pathogenic variant resulting in a heterozygous SHP2 p.Y62D missense mutation. She was found to have numerous, small, yellow-pink smooth papules that were histopathologically confirmed to be JXG. In understanding the common underlying pathogenetic dysregulation of the Ras/MAPK pathway in both NS and NF1, this report suggests a possible molecular association for why NS individuals may be predisposed to JXG.

Implications of mosaicism in variant interpretation: A case of a de novo homozygous NF1 variant.

Neurofibromatosis type 1 is a common multisystem autosomal dominant syndrome caused by pathogenic heterozygous variants in the neurofibromin gene (NF1). It is associated with a substantially increased cancer risk. Mosaicism for NF1 has been clinically well-established for "second hit" variants in skin lesions and tumor tissues. Here, we report on a 3-month-old boy with multiple café au lait macules (CAMs) and juvenile myelomonocytic leukemia (JMML) who was found to carry a previously established pathogenic NF1 variant (c.586+5G>A), as revealed by whole-exome sequencing. Surprisingly, however, this variant was detected in the homozygous state in the patient and was absent in the parents and siblings. Deep sequencing of this variant using blood, buccal swabs and skin samples was performed. As expected for an NF1 gene mutation promoting JMML, the variant was detected in 90.6% of the blood DNA reads, in sharp contrast to the mere 5% and 0.74% of reads in the saliva- and skin fibroblast-derived DNA, respectively. Our analysis, therefore, confirmed postzygotic origin of the variant followed by a mitotic event resulting in its homozygosity, although we could not differentiate between the possibilities of a gene conversion and mitotic crossover. Apparently de novo homozygous variants should trigger a careful investigation into mosaicism to achieve accurate interpretation.

Mutant SETBP1 enhances NRAS-driven MAPK pathway activation to promote aggressive leukemia.

Mutations in SET-binding protein 1 (SETBP1) are associated with poor outcomes in myeloid leukemias. In the Ras-driven leukemia, juvenile myelomonocytic leukemia, SETBP1 mutations are enriched in relapsed disease. While some mechanisms for SETBP1-driven oncogenesis have been established, it remains unclear how SETBP1 specifically modulates the biology of Ras-driven leukemias. In this study, we found that when co-expressed with Ras pathway mutations, SETBP1 promoted oncogenic transformation of murine bone marrow in vitro and aggressive myeloid leukemia in vivo. We demonstrate that SETBP1 enhances the NRAS gene expression signature, driving upregulation of mitogen-activated protein kinase (MAPK) signaling and downregulation of differentiation pathways. SETBP1 also enhances NRAS-driven phosphorylation of MAPK proteins. Cells expressing NRAS and SETBP1 are sensitive to inhibitors of the MAPK pathway, and treatment with the MEK inhibitor trametinib conferred a survival benefit in a mouse model of NRAS/SETBP1-mutant disease. Our data demonstrate that despite driving enhanced MAPK signaling, SETBP1-mutant cells remain susceptible to trametinib in vitro and in vivo, providing encouraging preclinical data for the use of trametinib in SETBP1-mutant disease.

Long non-coding RNAs as novel therapeutic targets in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50-60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

Myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes: a focused review.

Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are unique myeloid neoplasms, with overlapping features of MDS and MPN. They consist of four adult onset entities including chronic myelomonocytic leukemia (CMML), MDS/MPN-ring sideroblasts-thrombocytosis (MDS/MPN-RS-T), BCR-ABL1 negative atypical chronic myeloid leukemia (aCML) and MDS/MPN-unclassifiable (MDS/MPN-U); with juvenile myelomonocytic leukemia (JMML) being the only pediatric onset entity. Among these overlap neoplasms, CMML is the most frequent and is hallmarked by the presence of sustained peripheral blood monocytosis with recurrent mutations involving TET2 (60%), SRSF2 (50%) and ASXL1 (40%); with RAS pathway mutations and JAK2V617F being relatively enriched in proliferative CMML subtypes (WBC ≥13 × 109/L). CMML usually presents in the 7th decade of life, with a male preponderance and is associated with a median overall survival of <36 months. Adverse prognosticators in CMML include increasing age, high WBC, presence of circulating immature myeloid cells, anemia, thrombocytopenia and truncating ASXL1 mutations. While allogeneic stem cell transplantation remains the only curative option, given the late onset of this neoplasm and high frequency of comorbidities, most patients remain ineligible. Hypomethylating agents such as azacitidine, decitabine and oral decitabine/cedazuridine have been US FDA approved for the management of CMML, with overall response rates of 40-50% and complete remission rates of <20%. While these agents epigenetically restore hematopoiesis in a subset of responding patients, they do not impact mutational allele burdens and eventual disease progression to AML remains inevitable. Newer treatment modalities exploiting epigenetic, signaling and splicing abnormalities commonly seen in CMML are much needed.

CD14/16 monocyte profiling in juvenile myelomonocytic leukemia.

Monocyte subset analysis by flow cytometry has been shown to be a useful diagnostic tool in chronic myelomonocytic leukemia in adults. An increase in the classical monocyte fraction (CD14++/CD16-) greater than 94.0% of total monocytes is considered highly sensitive and specific in distinguishing chronic myelomonocytic leukemia from other myeloproliferative disorders. In a pilot study of juvenile myelomonocytic leukemia cases, we noted that CD14++/CD16- monocyte fraction was >95% in de novo juvenile myelomonocytic leukemia (JMML) with somatic PTPN11 mutations but normal in those with monosomy 7 or Noonan syndrome. Monocyte subgroup profiling by itself is not diagnostic of JMML but may distinguish molecular subgroups within JMML.

RAS Pathway Mutation Patterns in Patients With Juvenile Myelomonocytic Leukemia: A Developing Country Single-center Experience.

INTRODUCTION: Juvenile myelomonocytic leukemia (JMML) is a rare clonal myelodysplastic/myeloproliferative neoplasm of early childhood. Historically, it was difficult to diagnose clinically, as patients present with manifestations shared with other hematologic malignancies or viral infections. It is now clear that JMML is a disease of hyperactive RAS signaling. PATIENTS AND METHODS: We examined the bone marrow of 41 Egyptian children with JMML by direct sequencing for mutations in the RAS pathway genes. RESULTS: Mutations were detected in 33 (80%) of 41 patients. We identified 12 (29%) of 41 patients with PTPN11 mutation; 18 (44%) of 41 with RAS mutation; 9 (22%) of 41 with NRAS mutation; 9 (22%) of 41 with KRAS mutation; and 3 (7%) of 41 with CBL mutation. Eleven (92%) of the PTPN11 mutations were detected in exon 3 and 1 (8%) in exon 13. Seven of the NRAS mutations were in exon 2, and 2 were in exon 3. All KRAS mutations were in exon 2. The 3 cases with CBL mutation were homozygous mutations in exon 8. All the mutations detected in PTPN11, NRAS/KRAS, and the CBL genes were previously reported missense mutations in JMML. CONCLUSION: Our results demonstrate that Egyptian children diagnosed with JMML have high frequency of NRAS/KRAS mutations and lower frequency of PTPN11 mutations as compared with previous studies. The concept of mutually exclusive RAS pathway mutations was clearly observed in our patients. All cancer centers in our region should start implementing molecular diagnostic methods before confirming the diagnosis of JMML and before offering hematopoietic stem cell transplantation.

After 95 years, it's time to eRASe JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare clonal disorder of early childhood. Constitutive activation of the RAS pathway is the initial event in JMML. Around 90% of patients diagnosed with JMML carry a mutation in the PTPN11, NRAS, KRAS, NF1 or CBL genes. It has been demonstrated that after this first genetic event, an additional somatic mutation or epigenetic modification is involved in disease progression. The available genetic and clinical data have enabled researchers to establish relationships between JMML and several clinical conditions, including Noonan syndrome, Ras-associated lymphoproliferative disease, and Moyamoya disease. Despite scientific progress and the development of more effective treatments, JMML is still a deadly disease: the 5-year survival rate is ~50%. Here, we report on recent research having led to a better understanding of the genetic and molecular mechanisms involved in JMML.

A Case of Uveitis in a Patient With Juvenile Myelomonocytic Leukemia Successfully Treated With Adalimumab.

Patients with juvenile myelomonocytic leukemia due to germline CBL mutation (10% to 15%) may have a subacute course occasionally associated with autoimmune disorders, which may resemble RAS-associated autoimmune lymphoproliferative disorder. In both conditions, prognosis and standard treatment for autoimmune phenomena remain poorly understood. We report the case of a 7-year-old boy with juvenile myelomonocytic leukemia with severe steroid-dependent uveitis, who did not respond to several therapeutic attempts with immunosuppressant agents, including sirolimus, and was finally successfully treated with adalimumab. This case offers further insight into the management of autoimmune disorders in the context of predisposing genetic conditions.

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Diagnosis and treatment of juvenile myelomonocytic leukemia.

Objective: To investigate clinical features, diagnosis, treatment strategies and prognosis of juvenile myelomonocytic leukemia (JMML). Methods: The clinical data of 21 patients with JMML who were diagnosed in our hospital from January 2013 to May 2018 were retrospectively analyzed. Results: Among the 21 children with JMML, 16 were male and 5 were female. Out of the 21 children who were diagnosed with JMML, 7 were lost after treatment while the remaining 14 received A-3V chemotherapy regimen of South Korea. The effective response rate was 78.5%. The three-year overall survival (OS) rate and three-year disease-free survival (DFS) rate were (76.2 ± 14.8)% and (66.2 ± 14)%, respectively. Single factor analysis showed that PLT count ≤33×109/L, LDH level >500 U/L and HbF level >10% and chemotherapy only were the significant factors that lead to poor prognosis in children. Cox multivariate analysis showed that the choice of treatment options affected the prognosis of JMML children. By taking prognostic factors for long-term efficacy into account, patients with treatment strategy of chemotherapy alongside hematopoietic stem cell transplantation (HSCT) have a better prognosis. Conclusion: The PLT count, LDH level, HbF level and choice of treatment plan are important for the evaluation of prognosis for children with JMML. Although there is a lack of consistency in terms of donors but the A-3V scheme is relatively stable, so HSCT should be preferred for children with poor prognostic factors.

Essential role of PTPN11 mutation in enhanced haematopoietic differentiation potential of induced pluripotent stem cells of juvenile myelomonocytic leukaemia.

We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34+ and CD34+ CD45+ cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34+ cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34+ cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder.