Y. Lucy Liu

The genomic landscape of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 or CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and could therefore be candidates for experimental therapies. In addition, few molecular pathways aside from the RAS-MAPK pathway have been identified that could serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia to expand knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, splicing, Polycomb repressive complex 2 (PRC2) and transcription. Notably, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Childrens Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006).

Phase II/III trial of a pre-transplant farnesyl transferase inhibitor in juvenile myelomonocytic leukemia: a report from the Children's Oncology Group.

Juvenile myelomonocytic leukemia (JMML) is not durably responsive to chemotherapy, and approximately 50% of patients relapse after hematopoietic stem cell transplant (HSCT). Here we report the activity and acute toxicity of the farnesyl transferase inhibitor tipifarnib, the response rate to 13‐cis retinoic acid (CRA) in combination with cytoreductive chemotherapy, and survival following HSCT in children with JMML.

Timing of the loss of Pten protein determines disease severity in a mouse model of myeloid malignancy

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN). JMML leukemogenesis is linked to a hyperactivated RAS pathway, with driver mutations in the KRAS, NRAS, NF1, PTPN11, or CBL genes. Previous murine models demonstrated how those genes contributed to the selective hypersensitivity of JMML cells to granulocyte macrophage-colony-stimulating factor (GM-CSF), a unifying characteristic in the disease. However, it is unclear what causes the early death in children with JMML, because transformation to acute leukemia is rare. Here, we demonstrate that loss of Pten (phosphatase and tensin homolog) protein at postnatal day 8 in mice harboring Nf1 haploinsufficiency results in an aggressive MPN with death at a murine prepubertal age of 20 to 35 days (equivalent to an early juvenile age in JMML patients). The death in the mice was due to organ infiltration with monocytes/macrophages. There were elevated activities of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) in cells at physiological concentrations of GM-CSF. These were more pronounced in mice with Nf1 haploinsufficiency than in littermates with wild-type Nf1,but this model is insufficient to cause cells to be GM-CSF hypersensitive. This new model represents a murine MPN model with features of a pediatric unclassifiable mixed MDS/MPN and mimics many clinical manifestations of JMML in terms of age of onset, aggressiveness, and organ infiltration with monocytes/macrophages. Our data suggest that the timing of the loss of PTEN protein plays a critical role in determining the disease severity in myeloid malignancies. This model may be useful for studying the pathogenesis of pediatric diseases with alterations in the Ras pathway.

Corrigendum: The genomic landscape of juvenile myelomonocytic leukemia.

Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

PTEN is indispensable for cells to respond to MAPK inhibitors in myeloid leukemia

Constitutively activated MAPK and AKT signaling pathways are often found in solid tumors and leukemias. PTEN is one of the tumor suppressors that are frequently found deficient in patients with late-stage cancers or leukemias. In this study we demonstrate that a MAPK inhibitor, PD98059, inhibits both AKT and ERK phosphorylation in a human myeloid leukemia cell line (TF-1), but not in PTEN-deficient leukemia cells (TF-1a). Ectopic expression of wild-type PTEN in myeloid leukemia cells restored cytokine responsiveness at physiological concentrations of GM-CSF (<0.02 ng/mL) and significantly improved cell sensitivity to MAPK inhibitor. We also found that Early Growth Response 1 (EGR1) was constitutively over-expressed in cytokine-independent TF-1a cells, and ectopic expression of PTEN down-regulated EGR1 expression and restored dynamics of EGR1 expression in response to GM-CSF stimulation. Data from primary bone marrow cells from mice with Pten deletion further supports that PTEN is indispensible for myeloid leukemia cells in response to MAPK inhibitors. Finally, We demonstrate that the absence of EGR1 expression dynamics in response to GM-CSF stimulation is one of the mechanisms underlying drug resistance to MAPK inhibitors in leukemia cells with PTEN deficiency. Our data suggest a novel mechanism of PTEN in regulating expression of EGR1 in hematopoietic cells in response to cytokine stimulation. In conclusion, this study demonstrates that PTEN is dispensable for myeloid leukemia cells in response to MAPK inhibitors, and PTEN regulates EGR1 expression and contributes to the cytokine sensitivity in leukemia cells.

M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion

Uncovering the cellular and molecular mechanisms by which hematopoietic stem cell (HSC) self-renewal is regulated can lead to the development of new strategies for promoting ex vivo HSC expansion. Here, we report the discovery that alternative (M2)-polarized macrophages (M2-MΦs) promote, but classical (M1)-polarized macrophages (M1-MΦs) inhibit, the self-renewal and expansion of HSCs from mouse bone marrow (BM) in vitro. The opposite effects of M1-MΦs and M2-MΦs on mouse BM HSCs were attributed to their differential expression of nitric oxide synthase 2 (NOS2) and arginase 1 (Arg1), because genetic knockout of Nos2 and Arg1 or inhibition of these enzymes with a specific inhibitor abrogated the differential effects of M1-MΦs and M2-MΦs. The opposite effects of M1-MΦs and M2-MΦs on HSCs from human umbilical cord blood (hUCB) were also observed when hUCB CD34+ cells were cocultured with M1-MΦs and M2-MΦs generated from hUCB CD34- cells. Importantly, coculture of hUCB CD34+ cells with human M2-MΦs for 8 days resulted in 28.7- and 6.6-fold increases in the number of CD34+ cells and long-term SCID mice-repopulating cells, respectively, compared with uncultured hUCB CD34+ cells. Our findings could lead to the development of new strategies to promote ex vivo hUCB HSC expansion to improve the clinical utility and outcome of hUCB HSC transplantation and may provide new insights into the pathogenesis of hematological dysfunctions associated with infection and inflammation that can lead to differential macrophage polarization.

Erratum: Corrigendum: The genomic landscape of juvenile myelomonocytic leukemia

Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

Erratum: The genomic landscape of juvenile myelomonocytic leukemia (Nature Genetics (2015) 47 (1326-1333))

Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

Abstract 1070: Differential response dynamic of leukemia cells to GM-CSF and IL-3 stimulation in signal transduction pathways

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL GM-CSF and IL-3 regulate the survival, proliferation, differentiation, and functional activation of hematopoietic cells. GM-CSF also contributes to controlling the function of dendritic cells and T-cells. Aberrant function of GM-CSF has been linked to multiple diseases including juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia, rheumatoid arthritis, and alveolar proteinosis. GM-CSF and IL-3 share a common βc subunit on their receptor. Many studies have revealed that dys-regulation of Ras/PI3K/Akt and Ras/Raf/Mek/Erk pathways are responsible for hypersensitivities of GM-CSF and IL-3 in various diseases. However, few data have distinguished the difference of signal transduction pathways between the two cytokines. We previously reported that JMML cells are selectively hypersensitive to GM-CSF in vitro, while they show normal sensitivity to IL-3. Others documented that there was a time gap between CREB responded to GM-CSF and IL-3 stimulation. In the present study, we hypothesized that the signal transduction pathway of GM-CSF has differential response dynamic from that of IL-3. We first tested the CFU-GM growth pattern of a leukemia cell line, TF-1, in response to GM-CSF and IL-3 stimulation. We found that the GM-CSF dose-response curve was markedly shifted leftward, indicating that TF-1 cells were more sensitive to GM-CSF than to IL-3. This pattern is very similar to that observed in primary JMML cells. We next investigated the activities of the elements downstream in Ras pathway. After serum starving in medium with 0.5% BSA for 16 hours, TF-1 cells were stimulated with GM-CSF or IL-3 at a concentration range of 0.01-500pM. We found that the total protein levels of CREB were consistently unchanged. On the other hand, significant phosphorylation of CREB on serine 133 began to occur at concentrations of GM-CSF at 10pM. Significant phosphorylation of Erk was observed even at a concentration as low as 1 pM of GM-CSF. However, this phosphorylation pattern was not seen until 100pM of IL-3, and the level of phosphorylation was lower. The same dynamic, GM-CSF vs. IL-3, was also found in pAkt and pSTAT5. In conclusion, our data demonstrate that leukemia cells respond to GM-CSF and IL-3 stimulation in different dynamic patterns in signal transduction pathways. This indicates that the selective hypersensitivity to GM-CSF in some leukemia cells may be caused by aberrant elements in signal transduction pathways that are responsive to the low concentrations of GM-CSF stimulation. We raise a concern that any study related to responsiveness of GM-CSF or IL-3 stimulation should be interpreted cautiously; with specific attention paid to the concentration levels and the length of stimulation, in order to precisely characterize the function of GM-CSF and IL-3. This observed differential response dynamic might apply to a broader phenomenon in other cytokines or hormones in other tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1070. doi:1538-7445.AM2012-1070