Sustained fetal hematopoiesis causes juvenile death from leukemia: evidence from a dual-age-specific mouse model.

It is not clear whether disrupted age-specific hematopoiesis contributes to the complex manifestations in leukemia patients who carry identical mutations, particularly in pediatric and adult patients with similar clinical characteristics. By studying a dual-age-specific mouse model, we demonstrate that (1) loss of Pten during the fetal-to-adult hematopoiesis switch (hematopoiesis switch) causes sustained fetal hematopoiesis, resulting in death in juvenile leukemia; (2) myeloid-biased hematopoiesis in juvenile mice is associated with the sustained fetal properties of hematopoietic stem cells (HSCs); (3) the age specificity of juvenile myelomonocytic leukemia depends on the copy number of Pten and Nf1; (4) single-allelic Pten deletion during the hematopoiesis switch causes constitutive activation of MAPK in juvenile mice with Nf1 loss of heterozygosity (LOH); and (5) Nf1 LOH causes monocytosis in juvenile mice with Pten haploinsufficiency but does not cause lethality until adulthood. Our data suggest that 1 copy of Pten is sufficient to maintain an intact negative-feedback loop of the Akt pathway and HSC function in reconstitution, despite MAPK being constitutively activated in juvenile Pten+/ΔNf1LOH mice. However, 2 copies of Pten are required to maintain the integrity of the MAPK pathway in juvenile mice with Nf1 haploinsufficiency. Our data indicate that previous investigations of Pten function in wild-type mice may not reflect the impact of Pten loss in mice with Nf1 mutations or other genetic defects. We provide a proof of concept that disassociated age-specific hematopoiesis contributes to leukemogenesis and pediatric demise.

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.

Targeting tumor-associated carbohydrate antigens: a phase I study of a carbohydrate mimetic-peptide vaccine in stage IV breast cancer subjects.

Tumor-associated carbohydrate antigens (TACAs) support cell survival that could be interrupted by anti-TACA antibodies. Among TACAs that mediate cell survival signals are the neolactoseries antigen Lewis Y (LeY) and the ganglioside GD2. To induce sustained immunity against both LeY and GD2, we developed a carbohydrate mimicking peptide (CMP) as a surrogate pan-immunogen that mimics both. This CMP, referred to as P10s, is the N-terminal half of a peptide vaccine named P10s-PADRE, the C-terminal half of which (PADRE) is a Pan-T-cell epitope. A Phase I dose-escalation trial of P10s-PADRE plus adjuvant MONTANIDE™ ISA 51 VG was conducted in subjects with metastatic breast cancer to test 300 and 500 µg/injection in two cohorts of 3 subjects each. Doses of the P10s-PADRE vaccine were administered to research participants subcutaneously on weeks 1, 2, 3, 7 and 19. Antibody responses to P10s, GD2, and LeY were measured by ELISA. The P10s-PADRE vaccine induced antibodies specifically reactive with P10s, LeY and GD2 in all 6 subjects. Serum antibodies displayed Caspase-3-dependent apoptotic functionality against LeY or GD2 expressing breast cancer cell lines. Immunization with the P10s-PADRE vaccine was well-tolerated and induced functional antibodies, and the data suggest potential clinical benefit.

Managing Expectations in the Transition to Proof of Concept Studies.

BACKGROUND: As we move away from the traditional chemotherapy era to targeted therapy, the validity of old assessment paradigms associated with therapeutics are being raised in the context of immunotherapy. The old paradigm required elaborating on the toxicity assessment, with no expectation of efficacy in early phase trials. Safety data from Phase 1 and 2 studies with many immunotherapeutics show limited toxicities and draw attention to the need to demonstrate efficacy in the early evaluation of new agents. METHODS: Literature searches indicate that molecular oncology mechanistic-based agents are being linked with molecular disease status and clinical benefit. Biomarkers and other endpoints are being employed to accomplish this. Perspectives for a meaningful context of integrating biomarkers and clinical trial design are reviewed. RESULTS: The design and conduct of clinical trials have not been fully adjusted to the new era of personalized oncology, and so we are in transition. A part of this transition is the management of expectations and trial designs that need to be considered relative to preclinical experience in the development of therapeutics. For example, pathological complete response is now considered a surrogate marker for favorable prognosis in breast cancer patients who are treated in the neoadjuvant setting. This surrogate marker is tied to novel agents' mechanistic characteristics with no preclinical counterpart. CONCLUSION: The old paradigm considers patients equal with similar chances to respond to treatments, but the new paradigm considers patient's heterogeneity, a major fact that informs the design of clinical trials. By linking every treatment to a mechanism of action and to the presence of a specific biomarker, new trials are going to have more subjects who are likely to respond to the treatment.

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.

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.

MYC amplification in multiple marker chromosomes and EZH2 microdeletion in a man with acute myeloid leukemia.

The role of MYC and EZH2 in acute myeloid leukemia (AML) pathogenesis is poorly understood. Herein we present a case of AML with MYC amplification in marker chromosomes and a microdeletion of chromosome 7 below cytogenetic resolution. The karyotype of the patient's bone marrow aspirate showed three to five marker chromosomes in all dividing cells without other structural or numerical chromosomal abnormalities. Analysis by fluorescence in situ hybridization (FISH) with a probe specific for the human MYC gene revealed amplification of the oncogene localized to the marker chromosomes. Using whole genome single nucleotide polymorphism (SNP) microarray analysis, an approximately 4.4 Mb amplicon containing the MYC gene was identified with an estimated amplification of about 30 copies per leukemic cell and, thus, an average of about 8 copies per marker chromosome. A 6.4 Mb hemizygous microdeletion of chromosome 7 within band q36.1 was also found by SNP microarray analysis in a cellular-equivalent dosage of 50%. The microdeletion spans multiple genes, including EZH2, a gene with well-known cancer association. No mutation was found in the remaining EZH2 allele by next generation gene sequencing. The combination of MYC amplification and EZH2 deletion, which has not been described previously in AML, may suggest a synergistic role of the two oncogenes in the pathogenesis of the patient's acute leukemia.

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

BACKGROUND: 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. PROCEDURE: Eighty-five patients with newly diagnosed JMML were enrolled on AAML0122 between 2001 and 2006. Forty-seven consented to receive tipifarnib in a phase II window before proceeding to a phase III trial of CRA in combination with fludarabine and cytarabine followed by HSCT and maintenance CRA. Thirty-eight patients enrolled only in the phase III trial. RESULTS: Overall response rate was 51% after tipifarnib and 68% after fludarabine/cytarabine/CRA. Tipifarnib did not increase pre-transplant toxicities. Forty-six percent of the 44 patients who received protocol compliant HSCT relapsed. Five-year overall survival was 55 ± 11% and event-free survival was 41 ± 11%, with no significant difference between patients who did or did not receive tipifarnib. CONCLUSIONS: Administration of tipifarnib in the window setting followed by HSCT in patients with newly diagnosed JMML was safe and yielded a 51% initial response rate as a single agent, but failed to reduce relapse rates or improve long-term overall survival.

Moving a Carbohydrate Mimetic Peptide into the clinic.

Tumor-Associated Carbohydrate Antigens (TACAs) are broad-spectrum targets for immunotherapy. Immunization with Carbohydrate Mimetic Peptides (CMPs) is a strategy to induce broad-spectrum TACA-reactive antibodies hypothesized to interfere with cellular pathways involved in tumor cell survival. A Phase I study was conducted with a first-in-man CMP referred to as P10s, conjugated to the Pan T cell carrier PADRE, along with MONTANIDE(™) ISA 51 VG as adjuvant over a course of 5 immunizations. While designed as a safety and tolerability study, the potential for therapeutic impact was observed in a subject with metastatic lesions as evaluated before and after vaccine treatment. The subject received Vinorelbine and Trastuzumab (VT) for two months prior to study eligibility. PET scans showed partial response in the lungs and complete resolution of a previously enlarged subpectoral lymph node. Immunization with P10s vaccine resulted in responses to P10s, with serum and plasma antibodies reactive with and cytotoxic to human breast cancer cells in vitro, including the Trastuzumab-resistant HCC1954 cell line. However, the patient developed cystic masses in the brain parenchyma with no apparent evidence of metastases. The subject was switched to Docetaxel, Pertuzumab and Trastuzumab a year later, and her last PET scan showed a complete response in the lungs and lymph nodes. Incubation of cancer cells with a combination of vaccine-induced serum and docetaxel suggests that the induced antibodies sensitize tumor cells for more efficient killing upon administration of docetaxel. The data suggest that P10s-PADRE induces anti-tumor antibody response that in combination with chemotherapy can affect metastatic lesions in breast cancer patients.

Proteasome-associated autoinflammatory syndromes: advances in pathogeneses, clinical presentations, diagnosis, and management.

The disease spectrum currently known as the proteasome-associated autoinflammatory syndromes (PRAAS) was first described in 1939 in patients who presented with recurrent fevers beginning in infancy or early childhood, which were accompanied by nodular erythema, a pernio-like rash, and joint contractures. Since then, several syndromes, such as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome, Nakajo-Nishimura syndrome (NNS), joint contractures, muscle atrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP) syndrome, and Japanese autoinflammatory syndrome with lipodystrophy (JASL), have been used to categorize patients with diseases within the same spectrum. Recently, independent studies have identified mutations in the human proteasome subunit ß type 8 (PSMB8) gene, which result in a sustained inflammatory response in all syndromes. Further functional studies not only suggest a causative role of PSMB8 mutations but also imply that they represent one disease spectrum, referred to as PRAAS. In this paper, we review the clinical presentations and laboratory findings of PRAAS, as well as the most recent advances in pathogeneses, diagnosis, and treatment options for patients with diseases in this spectrum.

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 Children's 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).

Hallway gossip between Ras and PI3K pathways.

In this issue of Blood, Goodwin et al investigate the pathogenesis of juvenile myelomonocytic leukemia (JMML), demonstrating that mutant Shp2 induces granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity and that the p110δ subunit of phosphatidylinositol 3-kinase (PI3K) further promotes this dysregulation

Imatinib 800 mg daily induces deeper molecular responses than imatinib 400 mg daily: results of SWOG S0325, an intergroup randomized PHASE II trial in newly diagnosed chronic phase chronic myeloid leukaemia.

The standard dose of imatinib for newly diagnosed patients with chronic phase chronic myeloid leukaemia (CP-CML) is 400 mg daily (IM400), but the optimal dose is unknown. This randomized phase II study compared the rates of molecular, haematological and cytogenetic response to IM400 vs. imatinib 400 mg twice daily (IM800) in 153 adult patients with CP-CML. Dose adjustments for toxicity were flexible to maximize retention on study. Molecular response (MR) at 12 months was deeper in the IM800 arm (4-log reduction of BCR-ABL1 mRNA: 25% vs. 10% of patients, P = 0·038; 3-log reduction: 53% vs. 35%, P = 0·049). During the first 12 months BCR-ABL1 levels in the IM800 arm were an average 2·9-fold lower than in the IM400 arm (P = 0·010). Complete haematological response was similar, but complete cytogenetic response was higher with IM800 (85% vs. 67%, P = 0·040). Grade 3-4 toxicities were more common for IM800 (58% vs. 31%, P = 0·0007), and were most commonly haematological. Few patients have relapsed, progressed or died, but both progression-free (P = 0·048) and relapse-free (P = 0·031) survival were superior for IM800. In newly diagnosed CP-CML patients, IM800 induced deeper MRs than IM400, with a trend for improved progression-free and overall survival, but was associated with more severe toxicity.

PTEN transcript variants caused by illegitimate splicing in "aged" blood samples and EBV-transformed cell lines.

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Mutations occur in either heritable or sporadic fashion. Sequencing of cDNA from patients and normal individuals often reveals splicing variants (SVs) of PTEN, some of which are non-mutation related. To investigate whether these SVs were the result of illegitimate splicing (a general decrease of fidelity in splicing site selection in "aged" samples), we tested "aged" blood from individuals who had normal PTEN transcripts in their "fresh" mononuclear cells. Blood from 20 normal individuals was collected and split into two aliquots. Total RNA and DNA were extracted immediately ("fresh") and 48 h later ("aged"), respectively. Using RT-PCR, subcloning and sequencing, we found seven types of SVs. No mutation was detected in the related intron-exon flanking region in genomic DNA in either "fresh" or "aged" samples. Some of the SVs were also consistently present in both the "fresh" and "aged" EBV-transformed lymphoblastoid cells from six normal individuals. Western blot data indicated that the PTEN protein level (in full length) was not altered in the "fresh" EBV-transformed lymphoblastoid cells with SVs. In conclusion, our data demonstrate that PTEN illegitimate splicing often occurs in "aged" blood and EBV-transformed lymphoblastoid cells. Therefore, it is critical to note the time point of RNA extraction when investigating for PTEN aberrant transcripts. We hope that our data will increase awareness about the sample status, because gene expression data may be potentially flawed from "aged" samples, particularly when dealing with clinical samples.

Mutations in CBL occur frequently in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia is an aggressive myeloproliferative disorder characterized by malignant transformation in the hematopoietic stem cell compartment with proliferation of differentiated progeny. Seventy-five percent of patients harbor mutations in the NF1, NRAS, KRAS, or PTPN11 genes, which encode components of Ras signaling networks. Using single nucleotide polymorphism arrays, we identified a region of 11q isodisomy that contains the CBL gene in several JMML samples, and subsequently identified CBL mutations in 27 of 159 JMML samples. Thirteen of these mutations alter codon Y371. In this report, we also demonstrate that CBL and RAS/PTPN11 mutations were mutually exclusive in these patients. Moreover, the exclusivity of CBL mutations with respect to other Ras pathway-associated mutations indicates that CBL may have a role in deregulating this key pathway in JMML.

PTEN deficiency is a common defect in juvenile myelomonocytic leukemia.

The biological hallmark of juvenile myelomonocytic leukemia (JMML) is selective GM-CSF hypersensitivity. We hypothesized that PTEN protein deficiency might lead to insufficient negative growth signals to counter the hyperactive Ras signaling and therefore aid in the acceleration of the malignant transformation of JMML. In screening 34 JMML patients we found: (1) decreased PTEN protein in 67% of patients; (2) significantly lower PTEN mRNA levels in patients compared to controls (p<0.01); (3) a hypermethylated PTEN promoter in 77% of patients; and (4) constitutive-hyperactive Akt and MAPK in 55% and 73% of patients, respectively. These findings suggest that PTEN deficiency is very common in JMML and is in part due to hypermethylation of the PTEN gene promoter.