Stephen D. Nimer

Tet2 Loss Leads to Increased Hematopoietic Stem Cell Self-Renewal and Myeloid Transformation

Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.

Phase IIB Trial of Oral Midostaurin (PKC412), the FMS-Like Tyrosine Kinase 3 Receptor (FLT3) and Multi-Targeted Kinase Inhibitor, in Patients With Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome With Either Wild-Type or Mutated FLT3

PURPOSE Mutations leading to constitutive activation of the FMS-like tyrosine kinase 3 receptor (FLT3) occur in blasts of 30% of patients with acute myeloid leukemia (AML). Midostaurin (PKC412; N-benzoylstaurosporin) is a multitargeted tyrosine kinase inhibitor, with demonstrated activity in patients with AML/myelodysplastic syndrome (MDS) with FLT3 mutations. PATIENTS AND METHODS Ninety-five patients with AML or MDS with either wild-type (n = 60) or mutated (n = 35) FLT3 were randomly assigned to receive oral midostaurin at 50 or 100 mg twice daily. The drug was discontinued in the absence of response at 2 months, disease progression, or unacceptable toxicity. Response was defined as complete response, partial response (PR), hematologic improvement, or reduction in peripheral blood or bone marrow blasts by ≥ 50% (BR). RESULTS The rate of BR for the population in whom efficacy could be assessed (n = 92) was 71% in patients with FLT3-mutant and 42% in patients with FLT3 wild-type. One PR occurred in a patient with FLT3-mutant receiving the 100-mg dose regimen. Both doses were well-tolerated; there were no differences in toxicity or response rate according to the dose of midostaurin. CONCLUSION These results suggest that midostaurin has hematologic activity in both patients with FLT3-mutant and wild-type. The degree of clinical activity observed supports additional studies that combine midostaurin and other agents such as chemotherapy especially in FLT3-mutant AML.

Ifosfamide, carboplatin, and etoposide: a highly effective cytoreduction and peripheral-blood progenitor-cell mobilization regimen for transplant-eligible patients with non-Hodgkin's lymphoma.

PURPOSE To evaluate a chemotherapy regimen that consisted of ifosfamide administered as an infusion with bolus carboplatin, and etoposide (ICE) supported by granulocyte colony-stimulating factor (G-CSF) for cytoreduction and stem-cell mobilization in transplant-eligible patients with primary refractory or relapsed non-Hodgkins lymphoma (NHL). PATIENTS AND METHODS One hundred sixty-three transplant-eligible patients with relapsed or primary refractory NHL were treated from October 1993 to December 1997 with ICE chemotherapy at Memorial Sloan-Kettering Cancer Center. Administration of three cycles of ICE chemotherapy was planned at 2-week intervals. Peripheral-blood progenitor cells were collected after cycle 3, and all patients who achieved a partial response (PR) or complete response (CR) to ICE chemotherapy were eligible to proceed to transplantation. Event-free and overall survival, ICE-related toxicity, and the number of CD34(+) cells collected after treatment with ICE and G-CSF were evaluated. RESULTS All 163 patients were assessable for response, and there was no treatment-related mortality. A major response (CR/PR) was evident in 108 patients (66.3%); 89% of the responding patients underwent successful transplantation. Patient who underwent transplantation and achieved a CR to ICE had a superior overall survival to that of patients who achieved a PR (65% v 30%; P =.003). The median number of CD34(+) cells/kg collected was 8.4 x 10(6). The dose-limiting toxicity of ICE was hematologic, with 29.4% of patients developing grade 3/4 thrombocytopenia. There were minimal nonhematologic side effects. CONCLUSION ICE chemotherapy, with ifosfamide administered as a 24-hour infusion to decrease CNS side effects, and the substitution of carboplatin for cisplatin to minimize nephrotoxicity, is a very effective cytoreduction and mobilization regimen in patients with NHL. Furthermore, the quality of the clinical response to ICE predicts for posttransplant outcome.

L3MBTL1, a Histone-Methylation-Dependent Chromatin Lock

Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.

Heterodimeric JAK–STAT activation as a mechanism of persistence to JAK2 inhibitor therapy

The identification of somatic activating mutations in JAK2 (refs 1–4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cells sensitivity to Hsp90 inhibition.

The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF-/- mice have a profound reduction in the number of NK-T and NK cells. Purified MEF-/- NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNgamma. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.

Selective Depletion Of Cd8+ Cells For Prevention Of Graft-versus-host Disease After Bone Marrow Transplantation A Randomized Controlled Trial

We performed a prospective randomized, double-blind study to assess the efficacy of selective depletion of CD8+ bone marrow cells in preventing acute graft-versus-host disease (GVHD) in 38 patients undergoing HLA-identical sibling donor bone marrow transplantation for leukemia. All patients received CsA for GVHD prophylaxis. Nineteen patients received marrow depleted of CD8+ cells by ex vivo treatment with anti-leu2, an anti-CD8 mAb and complement; four patients had moderate (grade 1 or 2 acute GVHD) and the only patient who experienced grade 3 manifestations was a technical failure. The control group consisted of 19 patients who received unmodified bone marrow; one patient had grade 1, 4 patients had grade 2, and 10 had grade 3 or 4 acute GVHD. The actuarial incidence of grade > or = 2 acute GVHD was 20 +/- 20% in the CD8-depleted group compared with 80 +/- 18% in the controls (P = 0.004). Death in 5 of the control patients and the single patient in whom CD8 depletion was a technical failure was related to acute GVHD. Graft failure occurred in 2 patients in the CD8-depleted group and in none of the controls. Leukemic relapse occurred in 2 patients receiving CD8-depleted bone marrow and 2 patients in the control group. Seven patients receiving marrow depleted of CD8+ cells are alive and free of leukemia and 9 patients in the control group are alive, 7 of whom remain leukemia-free (P = 0.88). The 3-year actuarial leukemia-free survival is 37 +/- 22% of the CD8-depleted group and 36 +/- 22% for the control group. These results indicate that selective depletion of CD8+ cells from the bone marrow significantly reduces the incidence and severity of acute GVHD.

JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation.

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.

Normalization of pre-ASCT, FDG-PET imaging with second-line, non–cross-resistant, chemotherapy programs improves event-free survival in patients with Hodgkin lymphoma

We previously reported that remission duration < 1 year, extranodal disease, and B symptoms before salvage chemotherapy (SLT) can stratify relapsed or refractory Hodgkin lymphoma (HL) patients into favorable and unfavorable cohorts. In addition, pre-autologous stem cell transplant (ASCT) (18)FDG-PET response to SLT predicts outcome. This phase 2 study uses both pre-SLT prognostic factors and post-SLT FDG-PET response in a risk-adapted approach to improve PFS after high-dose radio-chemotherapy (HDT) and ASCT. The first SLT uses 2 cycles of ICE in a standard or augmented dose (ICE/aICE), followed by restaging FDG-PET scan. Patients with a negative scan received a transplant. If the FDG-PET scan remained positive, patients received 4 biweekly doses of gemcitabine, vinorelbine, and liposomal doxorubicin. Patients without evidence of disease progression proceeded to HDT/ASCT; those with progressive disease were study failures. At a median follow-up of 51 months, EFS analyzed by intent to treat as well as for transplanted patients is 70% and 79%, respectively. Patients transplanted with negative FDG-PET, pre-HDT/ASCT after 1 or 2 SLT programs, had an EFS of > 80%, versus 28.6% for patients with a positive scan (P < .001). This prospective study provides evidence that the goal of SLT in patients with Hodgkin lymphoma should be a negative FDG-PET scan before HDT/ASCT.