Juvenile Myelomonocytic Leukemia Patients Research Articles

Decitabine-based treatment strategy improved the outcome of HSCT in JMML: a retrospective cohort study.

Pre-HSCT disease control, suboptimal long-term prognosis, and a high recurrence incidence (RI) continue to pose significant challenges for hematopoietic stem cell transplantation (HSCT) in juvenile myelomonocytic leukemia (JMML) patients. This retrospective cohort study assessed the effectiveness of a decitabine (DAC)-based protocol in JMML patients undergoing HSCT. The pre-HSCT treatment includes initial and bridging treatment. The efficacy of DAC monotherapy versus DAC combined with cytotoxic chemotherapy(C-DAC) as initial treatment was compared, followed by DAC plus FLAG (fludarabine, cytarabine, and GCSF) as bridging treatment. The HSCT regimens were based on DAC, fludarabine, and busulfan. Post-HSCT, low-dose DAC was used as maintenance therapy. The study endpoints focused on pretransplantation simplified clinical response and post-HSCT survival. There were 109 patients, including 45 receiving DAC monotherapy and 64 undergoing C-DAC treatment. 106 patients completed bridging treatment. All patients were administered planned HSCT regimens and post-HSCT treatment. The initial treatment resulted in 88.1% of patients achieving clinical remission without a significant difference between the DAC and C-DAC groups (p=0.769). Clinical remission rates significantly improved following bridging treatment (p=0.019). The 5-year overall survival, leukemia-free survival, and RI were 92.2%, 88.4%, and 8.0%, respectively. A poor clinical response to pre-HSCT treatment emerged as a risk factor for OS (hazard ratio: 9.8, 95% CI: 2.3-41.1, p=0.002). Implementing a DAC-based administration strategy throughout the pre-HSCT period, during HSCT regimens, and in post-HSCT maintenance significantly reduced relapse and improved survival in JMML patients. Both DAC monotherapy and the DAC plus FLAG protocol proved effective as pre-HSCT treatments.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire.

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95% CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatment-related mortality (TRM) of 9.0% (95% CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95% CI: 65.7-82.4) and 66.4% (95% CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT ≥6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with 3 or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced, yet still substantial, relapse incidence. By integrating genetic information with clinical and hematologic features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit from novel therapeutic agents and post-transplant strategies.

Juvenile Myelomonocytic Leukemia – Experience from a Tertiary Care Hospital in Eastern India

Background: Juvenile myelomonocytic leukemia (JMML), previously known as juvenile chronic myeloid leukaemia, is a rare, unique, and aggressive myeloproliferative neoplasm of early childhood. Making a diagnosis of JMML is challenging because of the overlapping clinical and haematological features with other myeloproliferative neoplasms (MPN). However, some unique features like monocytosis, the absence of BCR-ABL translocation, and the presence of specific mutations (PTPN-11, K-RAS, N-RAS, CBL, or NF1) clinch the correct diagnosis.Methods: A prospective analysis of six JMML patients with variable clinical features treated with injection azacytidine as frontline therapy during the study period of 2 years. Results: The median age was 4.5 years with male:female ratio 2:4. Pallor and splenomegaly were the most common presenting signs. Four patients (66.67%) achieved complete remission (CR), two patients (33.33%) had partial remission (PR), and one patient (16.67%) had progressive disease (PD). The overall survival rate was 66.67% (four out of six), and the mortality rate was 33.33%.Conclusion: Azacitidine is an effective treatment option as upfront therapy for JMML, especially in resource poor developing countries.

Efficacy of Demethylated Drug Combine with Low Dose Chemotherapy in Juvenile Myelomonocytic Leukemia

Objection：Juvenile myelomonocytic leukemia (JMML) is a rare pediatric leukemia with a median survival time of less than 2 years. Currently, hematopoietic stem cell transplantation (HSCT) is the only possible cure of JMML. Most of the newly diagnosed pediatric patients with JMML have high tumor burden, and rapid disease progression, and may not tolerate HSCT. In the present study, we aimed to explore the effection of Demethylated drug in combination with low-dose chemotherapy on JMML patients before HSCT. Methods：A retrospective analysis of 16 JMML patients was performed from Jul 2017 to Nov 2021. All patients were given Demethylated drug in combination with low-dose chemotherapy, 10 patients decitabine at a dose of 20 mg/m2 for 5 days, supplemented with cytarabine (50-100 mg/m2×3~5 days), and /or etoposide (50 mg/m2×3~5 days), 6 patients Azacytidine at a dose of 75mg/m2 for 7 days combined with homoharringtonine 2mg/m2 for 5-7 days. Each treatment interval was 4 weeks, bridging to HSCT after three~four courses of treatment. Results：The median age of onset of the 16 JMML patients was 36 months (range 6-51 months), the male to female ratio was 14:2, and the median volume of the spleen at diagnosis was 357cm3 (range: 175-588) by Three-dimensional ultrasound of spleen. The median white blood cell (WBC) count was 39 (range 2-128) × 109/L, the median hemoglobin 84 (range 47-110) g/l, and the median platelet 36 (range 16-75) × 109/L，the median monocyte count was 8 (range 2-26) × 109/L at diagnosis, the median RDW-CV was 19 (15-30)%, the median RDW-SD 56 (45-77) fl, the median HbF 37(1-76)%. Chromosome was performed, of which 13 cases had normal karyotype. 2 cases had tirsomy 8, 1 case had structural abnormality in chrosome 9. Next-generation sequencing results showed that 11(68.8%) cases carried PTPN11 mutations, 6 (37.5%) cases NF1 mutation, 5 (31.3%) KRAS mutation, 4 (25%) NRAS mutation, 1 case without one of the Ras pathway genes. 8 cases (50%) had more than two types of canonical JMML mutations, among them 6 cases (75%) had PTPN11 and NF1 co-mutation. 10 cases had secondary gene mutations, 3 (3/10) cases JAK3 mutation, 2 (2/10) SETBP1 mutation, 2 (2/10) SH2B3 mutation, 2(2/10) ASXL1 mutation, 2 (2/10) ARID1A mutation, 1 (1/10) SRSF2 mutation. The median treatment course was three cycles (range: 1-6), and the response rate was 68.8% (11/16) after one cycle and 81.8% (9/11) after three cycles. One case achieved complete remission (CR) after four courses of therapy. The WBC and monocyte counts were significantly decreased after treatment (P<0.001), and the spleen volume showed a decreasing tendency. Eleven patients received HSCT, 9 cases were implanted successfully and 2 cases failed, of which 1 case was relieved by secondary transplantation and 2 patients died of grade IV graft-versus-host disease (GVHD) after transplantation. The median follow-up time was 31 months. The 2-year progression-free survival rate was 75.0±10.8%. Conclusions：Demethylated drug in combination with low-dose chemotherapy could reduce JMML patients' tumor burden, improve the general condition, and obtain a clinical response rate of 81.8% after 3 cycles therapy. Therefore, such a combination regimen could be used as a therapeutic option for JMML before HSCT.

Corelationship of Lymphocyte Subsets and Prognosis in Juvenile Myelomonocytic Leukemia

Objective: Juvenile Myelomonocytic Leukemia (JMML) is a rare hematopoietic stem cell malignant clonal disease in children and has the characteristics of MDS/MPN. Due to the poor response of JMML to chemotherapy, hematopoietic stem cell transplantation (HSCT) is currently the only cure method for JMML. Currently, there are some reports on the distribution of lymphocyte subsets in MDS. A significant lymphocytopenia was observed in MDS patients compared with similarly aged healthy people. Another study form Polish found patients with MDS have more CD3+T cells than healthy donors. But there are few related reports in JMML. The objective of this study is to analyze relationship between the distribution of lymphocyte subsets and prognosis in JMML patients, and explore new risk stratification indicatorsfor JMML personalized therapy. Method: This study retrospectively collected JMML patients clinical data from Institute of Hematology & Blood Diseases Hospital of CAMS during Jan 2008 and Nov 2022. All completed complete blood count and peripheral blood classification, fetal hemoglobin (HbF), granulocyte colony-stimulating factor (GM-CSF) high sensitivity, bone marrow morphology, immune typing, molecular biology, cytogenetics and other tests. We analyzed the relationship between distribution of lymphocyte and prognosis in these patients. Result: 96 newly diagnosed JMML patients were admitted to this study. The median age at diagnosis was 25 (2-82) months, and male to female was 3.8:1. 66 cases underwent lymphocyte subsets by flow cytometry. Median percentage of lymphocytes is 34.3%(1.8%-90.4%, normal range: 20%-40%), median percentage of CD3+T cells is 54.85%(10.3%-91%, normal range: 57%-88%), median percentage of CD3+CD4+ T cells is 30.95%(3.54%-53%, normal range: 27%-58%), median percentage of CD3+CD8+ T cells is 18.4%(2%-49.1%, normal range: 16%-45%), median percentage of NK cells is 13.95%(2.3%-50.3%, normal range: 3%-32%), and median percentage of B cells is 23.25%(4%-69.6%, normal range: 4%-18%). These cases were followed-up to Jan 2023, with a median follow-up time of 12 months (1-104 months). The overall survival (OS) rate was 52.67%, with a median OS time of 12 months (0-103 months). There were significant differences in the percentage of CD3+T cells (30.89% ± 9.46% vs 59.05% ± 10%, P<0.01) and CD3+CD4+T cells (14.87% ± 5.82% vs 35% ± 5.41%, P<0.01) between children with single NRAS mutation and compound mutations (≥2 classical five Ras pathway mutation ). In PTPN11 mutation patients, there is a significant positive correlation between OS and the percentage of lymphocytes (P=0.018). Conclusion: Our study shows differences in percentage of CD3+ T cells and CD3+CD4+ T cells between patients with NRAS mutation and compound mutations. Patients with PTPN11 mutation and higher percentage of lymphocytes will have better prognosis.

BH3 mimetics and azacitidine show synergistic effects on juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive hematopoietic disorder of infancy and early childhood driven by constitutively active RAS signaling and characterized by abnormal proliferation of the granulocytic-monocytic blood cell lineage. Most JMML patients require hematopoietic stem cell transplantation for cure, but the risk of relapse is high for some JMML subtypes. Azacitidine was shown to effectively reduce leukemic burden in a subset of JMML patients. However, variable response rates to azacitidine and the risk of drug resistance highlight the need for novel therapeutic approaches. Since RAS signaling is known to interfere with the intrinsic apoptosis pathway, we combined various BH3 mimetic drugs with azacitidine in our previously established patient-derived xenograft model. We demonstrate that JMML cells require both MCL-1 and BCL-XL for survival, and that these proteins can be effectively targeted by azacitidine and BH3 mimetic combination treatment. In vivo azacitidine acts via downregulation of antiapoptotic MCL-1 and upregulation of proapoptotic BH3-only. The combination of azacitidine with BCL-XL inhibition was superior to BCL-2 inhibition in eliminating JMML cells. Our findings emphasize the need to develop clinically applicable MCL-1 or BCL-XL inhibitors in order to enable novel combination therapies in JMML refractory to standard therapy.

Epigenetic Profiling of PTPN11 Mutant JMML Hematopoietic Stem and Progenitor Cells Reveals an Aberrant Histone Landscape.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

Combined Haploidentical Hematopoetic Stem Cell Transplantation and Liver Transplantation in a Pediatric Patient

Solid organ transplantation from the same donor is an established procedure for end-stage organ failure that developed after a previous hematopoietic stem cell transplantation (HSCT); however, it is rarely done in patients transplanted with unmanipulated haplo-HSCT. There are no pediatric reports regarding the long-term performance of organ transplantation after haplo-HSCT with post-transplant cyclophosphamide (PTCY).
 A juvenile myelomonocytic leukemia patient, who underwent unmanipulated haplo-HSCT with PTCY from her mother at the age of 3 years, developed chronic liver graft versus host disease (GvHD) which was refractory to specific GvHD treatment. Liver transplantation (LT) from her mother (the donor of her haplo-HSCT) was decided as the next line of treatment.
 LT was performed on day 540 post-HSCT, and the donor's left lateral segment was appropriately removed and attached to the recipient. The symptoms of GvHD completely regressed in a month. The patient died on day 121 after LT, because of a possible hepato-pulmonary syndrome.
 Organ failure can develop after allo-HSCT secondary to GvHD and therefore performing HSCT from a haplo-donor may be superior to a matched unrelated donor in terms of subsequent organ transplantation for organ failure.

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only “canonical” mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Juvenile myelomonocytic leukemia: A national analysis of demographic features.

e19086 Background: Juvenile Myelomonocytic Leukemia (JMML) is a rare hematological cancer originating in the bone marrow. It is a type of myelodysplastic syndrome (MDS) characterized by abnormal production of myeloid progenitor cells and monocytes. This malignancy is predominant in males, has a median age of diagnosis of 2 years, and carries a poor prognosis. It only accounts for 1% of all pediatric leukemias but constitutes 20-40% of pediatric MDS cases, making it the most common MDS subtype in children. Hematopoietic stem cell transplant (HSCT) is the only known cure for JMML, but its efficacy is just 50%. Without treatment, patients survive for a median of 10-12 months. Given the rarity of JMML, an analysis of demographic trends could offer valuable epidemiological insight. The National Cancer Database (NCDB) database was used to examine major demographic features of JMML. Methods: A retrospective analysis of all patients diagnosed with JMML between 2004 – 2020 in the NCDB (N = 242) was performed. Demographic factors including sex, race, Hispanic ethnicity, household income status, insurance status, urban/rural status, and Charlson-Deyo score were examined by descriptive statistics. Treatment statistics were also analyzed. Linear regression was used to determine incidence trends. Results: A total of 242 patients were identified with a histologically-confirmed diagnosis of JMML. Most patients were male (67.4%). The average age of diagnosis was 2 years (SD = 3.69, range = 0-33). The average incidence was 14.2 patients diagnosed per year (SD = 3.7, range = 9 – 21, R2 = 0.131). 93.8% had a Charlson-Deyo comorbidity score of 0. The majority of patients were White (74.4%), followed by Black (11.6%). 16.1% of patients were of Hispanic ethnicity. The distribution of median household income was 27.4% in the top quartile, 26.9% in the second, 26.9% in the third, and 18.7% in the fourth. Most patients were either privately insured (49.6%) or insured by Medicaid (42.6%). 75.5% of patients lived in a county with 250,000 people or more. Most patients received treatment (84.6%), while some only received active surveillance (8.7%) or no treatment (6.7%). Conclusions: This is the first NCDB analysis describing the demographic trends of JMML. Patients were twice as likely to be male than female. They were most likely to be White, with an average age of 2 years. This data reflects the expected age group and sex/race predominance described in the literature. The proportion of Hispanic JMML patients was comparable to Hispanics in the general US population. JMML appeared to be nearly evenly distributed among the top three household income quartiles. The lowest income quartile and rural inhabitants were least affected by this disease, which may indicate underdiagnosis in these populations. Further studies are needed to better define the relevance of these individual factors in the diagnosis, treatment, and survival of patients with JMML.

Germline Neurofibromin 1 mutation enhances the anti-tumour immune response and decreases juvenile myelomonocytic leukaemia tumourigenicity.

Juvenile myelomonocytic leukaemia (JMML) is an aggressive paediatric leukaemia characterized by mutations in five canonical RAS pathway genes, including the NF1 gene. JMML is driven by germline NF1 gene mutations, with additional somatic aberrations resulting in the NF1 biallelic inactivation, leading to disease progression. Germline mutations in the NF1 gene alone primarily cause benign neurofibromatosis type 1 (NF1) tumours rather than malignant JMML, yet the underlying mechanism remains unclear. Here, we demonstrate that with reduced NF1 gene dose, immune cells are promoted in anti-tumour immune response. Comparing the biological properties of JMML and NF1 patients, we found that not only JMML but also NF1 patients driven by NF1 mutations could increase monocytes generation. But monocytes cannot further malignant development in NF1 patients. Utilizing haematopoietic and macrophage differentiation from iPSCs, we revealed that NF1 mutations or knockout (KO) recapitulated the classical haematopoietic pathological features of JMML with reduced NF1 gene dose. NF1 mutations or KO promoted the proliferation and immune function of NK cells and iMacs derived from iPSCs. Moreover, NF1-mutated iNKs had a high capacity to kill NF1-KO iMacs. NF1-mutated or KO iNKs administration delayed leukaemia progression in a xenograft animal model. Our findings demonstrate that germline NF1 mutations alone cannot directly drive JMML development and suggest a potential cell immunotherapy for JMML patients.

Potential clinical use of azacitidine and MEK inhibitor combination therapy in PTPN11-mutated juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of childhood. The molecular hallmark of JMML is hyperactivation of the Ras/MAPK pathway with the most common cause being mutations in the gene PTPN11, encoding the protein tyrosine phosphatase SHP2. Current strategies for treating JMML include using the hypomethylating agent, 5-azacitidine (5-Aza) or MEK inhibitors trametinib and PD0325901 (PD-901), but none of these are curative as monotherapy. Utilizing an Shp2E76K/+ murine model of JMML, we show that the combination of 5-Aza and PD-901 modulates several hematologic abnormalities often seen in JMML patients, in part by reducing the burden of leukemic hematopoietic stem and progenitor cells (HSC/Ps). The reduced JMML features in drug-treated mice were associated with a decrease in p-MEK and p-ERK levels in Shp2E76K/+ mice treated with the combination of 5-Aza and PD-901. RNA-sequencing analysis revealed a reduction in several RAS and MAPK signaling-related genes. Additionally, a decrease in the expression of genes associated with inflammation and myeloid leukemia was also observed in Shp2E76K/+ mice treated with the combination of the two drugs. Finally, we report two patients with JMML and PTPN11 mutations treated with 5-Aza, trametinib, and chemotherapy who experienced a clinical response because of the combination treatment.

Targeting Arg-1 and PD-L1 in M2-Tumor Associated Macrophages Impairs Juvenile Myelomonocytic Leukemia (JMML) Cell Proliferation and Migration

Background and Hypothesis: Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. They are largely characterized into M1 or M2 types. TAMs express an anti-inflammatory M2-like phenotype, promote tumor progression. However, the role of M2-TAMs in driving disease pathogenesis in patients with Juvenile myelomonocytic leukemia (JMML), a rare form of pediatric leukemia driven to a large extent by mutations in the PTPN11 gene, which encodes the phosphatase SHP2 is unclear. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-TAMs express higher levels of arginase-1 (Arg-1) and PD-L1, which may contribute to the local suppression of immune responses and support the development of JMML.
 Methods: To study how alterations in M1/M2 macrophages contribute to JMML development, we utilized a mouse model bearing Shp2E76K mutation (Ptpn11E76K/+) which manifests the cardinal features of human JMML. We hypothesized that Shp2E76K/+ mutations enhance the function of bone marrow derived macrophages (BMDMs), including M2-TAMs and contribute to T-cell suppression.
 Results: Our analysis of the bulk RNA-sequence data from 90 JMML patients showed increase in the expression of Arg-1 and PD-1. Furthermore, single cell RNA-seq analysis of macrophages from 4 JMML patients revealed higher expression of M2-macrophage markers/genes. Our results show that in M2-TAMs, Arg-1 and PD-L1 levels are elevated in BM and spleens of Shp2E76K/+ mice compared to WT. Moreover, M2-TAMs, Arg-1 and PD-L1 levels were also higher in BMDMs derived from Shp2E76K/+ mice compared to WT. The BMDMs from Shp2E76K/+ mice have greater proliferation and migration potential compared to WT BMDMs, which was significantly reduced by inhibiting the function of Arg-1 and PD-L1.
 Conclusion: Our results show that M2-TAMs, arginase-1, and PD-L1 create a pro-tumor microenvironment, which likely contributes to the growth of JMML cells. Inhibition of Arg-1 and PD-L1 is a novel therapeutic approach to treat patients with JMML.

Genetic characteristics and survival analysis of 27 cases of juvenile myelomonocytic leukemia

Objective: To investigate the genetic and genomic profiling of juvenile myelomonocytic leukemia (JMML) and factors affecting its survival rate. Methods: Clinical characteristics, cytogenetics, molecular biology results and survival status of children with 27 JMML cases admitted to the Hematology Department of Children's Hospital, Capital Institute of Pediatrics from December 2012 to December 2021 were analyzed retrospectively, and the outcomes of the children were followed up. Kaplan-Meier method was used for survival analysis. Univariate analysis was used for analyzing factors affecting the overall survival (OS) rates of patients who received hematopoietic stem cell transplantation (HSCT). Log-Rank test was used for comparison of survival curves. Results: Among 27 JMML cases, there were 11 males and 16 females. The age of disease onset was 28 (11,52) months. There are 20 cases of normal karyotype, 4 cases of monosomy 7, 1 case of trisomy 8,1 case of 11q23 rearrangement and 1 case of complex karyotype. A total of 39 somatic mutations were detected.Those involved in RAS signal pathway were the highest (64%(25/39)), among which PTPN11 mutation was the most frequent (44% (11/25)). A total of 17 cases (63%) received HSCT, 8 cases (30%) did not receive HSCT, and 2 cases (7%) lost follow-up. For children receiving transplantation, the follow-up time after transplantation was 47 (11,57) months. The 1-year OS rate of high-risk transplantation group (17 cases) and high-risk non transplantation group (6 cases) was (88±8)% and (50±20)% respectively, with a statistically significant difference (χ2=5.01, P=0.025). The 5-year OS rate of the high-risk transplantation group was (75±11)%. The survival time of those who relapsed or progressed to acute myeloid leukemia after transplantation was significantly shorter than that of those who did not relapse (χ2=6.80, P=0.009). The OS rate of patients with or without PTPN11 mutation was (81±12) % and (67±19)% respectively (χ2=0.85, P=0.356). Conclusions: The main pathogenesis involved in JMML is gene mutation related to RAS signaling pathway, and the most common driver gene of mutation is PTPN11. Allogeneic HSCT can significantly improve the survival rate of high-risk JMML patients. The recurrence or progression after transplantation was related to poor prognosis.

Novel Humanized Loss-of-Function NF1 Mouse Model of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric cancer characterized by splenomegaly, excessive accumulation of monocytes/macrophages and myelodysplastic features. Mutations in the RAS pathway are the major drivers, with NF1 loss-of-function (NF1LOF) mutations found in up to 20% of patients who experience particularly dismal outcomes. Neurofibromin1 (NF1) gene negatively regulates RAS/MAPK/PI3K pathways by converting active Ras-GTP to inactive Ras-GDP to control proliferation/differentiation of immature myeloid cells (Side et al., 1998). In JMML patients, NF1mutations results in either termination of protein translation or translation of truncated, non-functional protein that upregulates the RAS pathway. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ~50% of patients. Therefore, there is an urgent need for novel therapeutic strategies. However, to date, clarifying the mechanism of disease development has been challenging due to low patients’ sample availability and lack of reliable humanized animal models which are the gold standard for studying rare diseases. While there has been some success in developing transgenic (Nf1LOF) and xenograft (Ptpn11, Nras, and Kras mutated) murine models of JMML (Liu et al., 2016, Krombholz et al., 2016, Yoshimi et al., 2017), there is still a need for a reliable preclinical humanized model. We used CRISPR/Cas9 to create NF1LOF in healthy juvenile hematopoietic stem and progenitor cells (HSPCs). We knocked out (KO) the human NF1 gene in HSPCs from healthy donor umbilical cord blood (UCBs) by designing single guide RNAs targeting NF1 Exon 2 to mimic the loss of protein function found in cells of JMML patients. We consistently obtained KO scores of ~85% in modified UCB HSPCs (Fig.1A). We confirmed the loss of full-length NF1 protein and presence of a truncated protein in the modified cells by western blot. Additionally, we observed significant hypersensitivity of NF1-KO cells to GM-CSF in colony-forming unit assays in vitro, a unique characteristic feature of JMML patient cells (Emanuel et al., 1991). Thereafter, we assessed the ability of NF1-KO HSPCs to initiate leukemia in humanized immunodeficient NSG-SGM3 neonates. We performed intrahepatic transplantation of the NF1LOF UCBs at post-natal days 2-4. We observed signs of leukemia (including weight loss, weakness, monocytosis in blood, anemia, enlarged spleen and liver) in all mice transplanted with modified NF1-KO cells, with a median survival of 5 weeks (Fig.1B). Significant splenomegaly was observed in all leukemic mice, along with the abrogation of lymphocyte production and an accumulation of myeloid cells (CD33+, CD13+, CD33+CD13+, CD14+, CD16+ and CD14+CD16+) and NK cells (CD56+/BrightCD16+) in the bone marrow and spleen of all leukemic mice, as observed by flow cytometry. This phenotype is consistent with that observed in patient-derived xenograft models of JMML (Krombholz et al., 2016). Extensive leukemia infiltration was also observed in liver and lungs, similar to what is observed in JMML patients. We are currently assessing the long-term leukemia initiation potential of CRISPR-modified NF1-KO cells through secondary transplants. In conclusion, we have successfully developed a novel humanized mouse model of JMML carrying NF1LOF mutation using CRISPR/Cas9 genome editing technology, with features (splenomegaly, monocytosis, and GM-CSF hypersensitivity) that have been reported as unique characteristics of this disease. This provides a new tool to investigate mechanisms of disease development, epigenetic dysregulation pathways, and to ultimately identify potential targets and test therapeutic interventions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LNK/SH2B3 As a Novel Driver in Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative disorder in toddlers. Ninety-five percent of patients have mutations detected in the Ras/MAPK signaling pathway including CBL, KRAS, NF1, NRAS, RRAS, RRAS2, and PTPN11. Oncogenic fusion proteins leading to hyperactive Ras signaling and other mutations in genes encoding for proteins upstream the Ras pathway have also been described. One of these upstream proteins is LNK which is encoded by SH2B3. Loss of function mutations in SH2B3 result in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of JAK/STAT signaling. Mutations in SH2B3 have been detected in a variety of hematologic malignancies, specifically in myeloproliferative neoplasms in adults. We previously identified secondary mutations in SH2B3 in JMML. Here we report a case series of four JMML patients with initiating mutations in SH2B3. We also show that SH2B3 mutant induced pluripotent stem cell (iPSC)-derived JMML like hematopoietic progenitor cells (HPC) are sensitive to JAK inhibitors including ruxolitinib. Patients and Methods Genomic DNA from peripheral blood, bone marrow or buccal swabs was extracted using standard protocols. DNA samples were sequenced using a custom amplicon-based targeted sequencing approach. Ficoll-purified mononuclear cells from bone marrow of JMML patients and control samples were reprogrammed by using Sendai virus expressing doxycycline-regulated OCT4, KLF4, MYC, and SOX2. Control and JMML iPSCs were differentiated to HPCs by culturing in serum-free media with sequential combinations of growth factors to support multipotent hematopoietic progenitor formation. The half-maximal inhibitory concentration (IC50) for each kinase inhibitor was determined by performing Cell Titer Glo assays (Promega). Results Clinical characteristics of patients with SH2B3 mutant JMML described in this study are highlighted in Table 1. iPSC-derived JMML-like HPCs with different mutational backgrounds were analyzed in cell proliferation assays after exposure to multiple different JAK inhibitors (ruxolitinib, momelotinib, tofacitinib). HPCs with alterations in SH2B3 were more sensitive to chemical JAK inhibition compared to HPCs not harboring mutations in SH2B3. This finding was observed for all JAK inhibitors but was most prominent with ruxolitinib (Figure 1). Patient 3 received ruxolitinib monotherapy after being refractory to 6-mercaptopurine. Treatment resulted in resolution of splenomegaly but no reduction in the variant allele frequency of SH2B3. The patient is now alive more than one year after hematopoietic stem cell transplantation (HSCT). Discussion In a previous study of 100 patients with JMML we first identified seven patients with secondary SH2B3 mutations. Here, we report four additional patients with JMML that appear to be driven by initiating mutations in SH2B3 (Table 1). The mutations identified here result in a truncated protein (patient 1, 3 and 4) or affect the SH2 domain (patient 2). Interestingly, copy neutral loss of heterozygosity of SH2B3 in patient 1, also known as uniparental isodisomy, is a mechanism that has been observed in other genes (e.g. CBL and NF1) in JMML. Since in vitro data showed that loss of LNK results in increased JAK/STAT signaling, we hypothesized that this cohort of patients may benefit from JAK inhibitor therapy. Our data from iPSC-derived JMML like HPCs shows, that those cells with an additional SH2B3 mutation are more sensitive to JAK inhibitors, specifically ruxolitinib, a JAK1/2 inhibitor that is already approved or under investigation in multiple clinical trials. In summary, we expand the list of initiating mutations in JMML to include SH2B3 and raise the possibility of targeting the JAK pathway in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omics Profiling of JMML HSPCs Reveals Onco-Fetal Reprogramming and Identifies Novel Prognostic Biomarkers and Therapeutic Targets in High-Risk JMML

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of early childhood, which is characterized by highly heterogenous clinical outcomes ranging from spontaneous resolution to early relapse after hematopoietic stem cell transplantation (HSCT). Established clinical risk factors as well as characteristic mutational patterns do not fully explain this heterogeneity. Recently, we and others identified DNA methylation subgroups in JMML, which were shown to be the only significant factor for predicting overall survival (Schönung et al., Clinical Cancer Research 2021). Together, these findings suggest a functional role for DNA methylation in the molecular pathogenesis of JMML. To elucidate the impact of aberrant DNA methylation on pathogenesis and disease progression, we leveraged a multi-modal molecular analysis approach. We integrated flow cytometry data with single-cell RNA-seq and ultra-low input whole-genome bisulfite sequencing (WGBS) data in order to dissect the hematopoietic system across patients from all epigenetic subgroups (n=8). With this approach, we aimed to identify novel candidate prognostic biomarkers and therapeutic targets for high-risk JMML patients. We observed conservation of epigenetic subgroups already in the hematopoietic stem and progenitor cell (HSPC) compartment, suggesting that differential methylation might be key to understand pathogenesis and course of the disease in JMML. In addition, single-cell transcriptomics revealed disease-specific aberrations to be most pronounced in the HSPC compartment relative to all other hematopoietic cell types. Consequently, we focused our work on JMML HSPCs, which revealed a high degree of immunophenotypic, transcriptomic and methylomic heterogeneity across JMML risk groups. This included the expression of proto-oncogenes belonging to the AP-1 family, CTNNB1 and MECOM (EVI1). Furthermore, we observed divergent lineage priming across JMML subgroups which affected lymphoid, myeloid and erythroid differentiation programs. Remarkably, subgroup-specific aberrations comprised upregulation of fetal developmental programs in HSPCs from high-risk JMML patients. This is of particular interest regarding the cell-of-origin in JMML, since mutations activating the RAS signaling pathway have been identified in newborn blood samples from children who later developed JMML (Behnert et al., Leukemia 2022). Comparing the methylome data of HSPCs from JMML patients with HSPCs from healthy individuals at different developmental stages (fetal, perinatal, juvenile and adult) suggested progressive epigenomic reprogramming of leukemic HSPCs towards high-risk JMML. As a consequence, we observed signatures of both accelerated aging and aberrant activation of onco-fetal programs. This included the downregulation of postnatal HSPC markers such as AVP and vice versa the upregulation of prenatal markers such as HMGA2 in high-risk HSPCs, which was associated with differential methylation of these genes. Moreover, binding motifs of central hematopoietic and developmental transcription factors such as RUNX1, FLI1, CDX4, and several HOX genes were not only enriched in differentially methylated regions but were also found to be differentially methylated themselves. Systematic comparison of JMML HSPCs with their healthy nearest normal counterparts revealed disease-specific expression of several cell surface markers. The expression patterns appeared to be regulated in a subgroup-specific manner, which nominates these factors as novel prognostic biomarkers in JMML. Moreover, we identified surface markers that were not only differentially methylated but also known drug targets in other entities, which is why we selected these genes as high confidence candidates for further functional validation. In a preclinical patient-derived xenograft (PDX) mouse model, targeted treatment with a therapeutic antibody against one of these candidates efficiently depleted high-risk JMML HSPCs and improved survival of JMML-PDX mice. In conclusion, the personalized molecular analysis of JMML HSPCs disentangled disease-specific heterogeneity and provided novel insights into tumorigenesis. Subgroup-specific epigenetic dysregulation included onco-fetal reprogramming of JMML HSPCs and revealed novel prognostic biomarkers and promising novel drug targets specifically for high-risk JMML.

Aurora Kinase B/C Inhibitor GSK1070916 Specifically Targets Juvenile Myelomonocytic Leukemia Cells with SHP2(PTPN11) Mutation

Juvenile myelomonocytic leukemia (JMML) is a rare and fatal early childhood hematological malignancy. Somatic SHP2 mutation accounts for 30-40% of JMML, with poor prognosis and lack of effective therapy methods. Aurora kinases play important roles in chromosome alignment, segregation and cell division during mitosis. Malignant hematopoietic cell diseases, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as chronic myeloid leukemia (CML), aberrantly express Aurora kinase A or B, which predict a poor overall prognosis. Aurora kinase represents a promising therapeutic target. However, to date there is no study on the relationship of aurora kinase and SHP2 mutant JMML. HCD57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2/D61Y and SHP2/E76K transformed HCD57 cells through retroviral transfection and selection. The two generated transformed cell lines (HCD57-SHP2/D61Y, HCD57-SHP2/E76K) were dependent on mutated-SHP2-mediated signaling. Based on these three cell lines, we screened 12 inhibitors, including aurora kinase A inhibitors, aurora kinase B inhibitors and pan-aurora kinase inhibitors. The selectivity and efficacy of the lead drugs were further evaluated on SHP2 mutant HCD57 cells and patient-derived PBMC cells by cell viability, flow cytometry, shRNA mediated gene silencing, western blotting and colony formation assays. Previous studies have shown that our established SHP2-mutant HCD57 cell line is a leukemia cell line representing the molecular signature of signaling pathways in SHP2 mutant JMML. The Aurora kinase B/C inhibitor GSK1070916 selectively inhibited SHP2-mutant HCD57 cells through drug screening. Cell viability assay demonstrated a dose-dependent inhibition pattern of HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells and had no significant inhibition effect on parental HCD57 cells. GSK1070916 induced cell cycle arrest at G2/M phase and cell apoptosis in HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells. Although HCD57 cells were also arrested in G2/M phase after GSK1070916C treatment, the cells did not show significant apoptosis. Western blot analysis showed that the protein level of anti-apoptotic protein Bcl-xL demonstrated a time-dependent increase, while caspase-3 and PARP proteins had no obvious cleavage in HCD57 cells after treatment of GSK1070916. However, in HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells, protein level of Bcl-xL was not significantly increased, while the cleaved form of caspase-3 and PARP significantly increased. This result demonstrated that SHP2-mutant cells were less capable in generating pro-survival signal and was sensitive to the inhibition of AURKB. Furthermore, the phenotype induced through silencing Aurkb by shRNA was similar to the inhibition effect of GSK1070916. We further investigated the ex vivo inhibitory effect of GSK1070916 on JMML patient-derived PBMC cells (mutation type: SHP2/E76A). The colony-forming ability of primary JMML cells was completely inhibited under treatment of GSK1070916 (500 nM). In conclusion, aurora kinase B can be used as a promising therapeutic target for SHP2-mutant JMML patients, and the aurora kinase B/C inhibitor GSK1070916 could be a candidate for the treatment of JMML.