Tobias Neff

Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer.

Enhancer of zeste homolog2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized alpha-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2/EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte/macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2/EED complex, we pharmacologically modulate an epigenetic “writer” and suppress PRC2-dependent cancer cell growth.

Genetic and Pharmacologic Inhibition of β-Catenin Targets Imatinib Resistant Leukemia Stem Cells in CML

A key characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Genetic deletion of β-catenin during fetal HSC development leads to impairment of self-renewal while β-catenin is dispensable in fully developed adult HSCs. Whether β-catenin is required for maintenance of fully developed CML leukemia stem cells (LSCs) is unknown. Here, we use a conditional mouse model to show that deletion of β-catenin after CML initiation does not lead to a significant increase in survival. However, deletion of β-catenin synergizes with imatinib (IM) to delay disease recurrence after imatinib discontinuation and to abrogate CML stem cells. These effects can be mimicked by pharmacologic inhibition of β-catenin via modulation of prostaglandin signaling. Treatment with the cyclooxygenase inhibitor indomethacin reduces β-catenin levels and leads to a reduction in LSCs. In conclusion, inhibiting β-catenin by genetic inactivation or pharmacologic modulation is an effective combination therapy with imatinib and targets CML stem cells.

Polycomb repressive complex 2 is required for MLL-AF9 leukemia

A growing body of data suggests the importance of epigenetic mechanisms in cancer. Polycomb repressive complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers. However, its role in cancer development and progression remains unclear. We used conditional alleles for the PRC2 components enhancer of zeste 2 (Ezh2) and embryonic ectoderm development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared with wild-type leukemia, Ezh2-null MLL-AF9–mediated acute myeloid leukemia (AML) failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML, but plays a role in leukemia progression. Genome-wide analyses of PRC2-mediated trimethylation of histone 3 demonstrated locus-specific persistence of H3K27me3 despite inactivation of Ezh2, suggesting partial compensation by Ezh1. In contrast, inactivation of the essential PRC2 gene, Eed, led to complete ablation of PRC2 function, which was incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared with Ezh2-null leukemic cells, including down-regulation of Myc target genes and up-regulation of PRC2 targets. Manipulating PRC2 function may be of therapeutic benefit in AML.

Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

Extended Survival of Glioblastoma Patients After Chemoprotective HSC Gene Therapy

Gene therapy using P140K-modified hematopoietic progenitor cells is chemoprotective, enabling glioblastoma patients to withstand myelotoxic doses of chemotherapy. Arming Blood Stem Cells to Fight Cancer The toxic effects of chemotherapy (chemotoxicity) on blood and bone marrow cells of cancer patients can be a significant barrier to treating tumors. Delivery of a gene that can protect bone marrow stem and progenitor cells from chemotoxicity could overcome this barrier. In a new study by Adair et al., patients with chemotherapy-resistant brain tumors with very poor chances of survival were given a transplant with their own bone marrow hematopoietic stem cells after the cells had been modified with a gene that protects these cells from chemotherapy. After the bone marrow transplant, patients were then given dose-intensified chemotherapy. Adair et al. report that the patients were able to tolerate these chemotherapy doses better after transplant of the gene-modified bone marrow stem cells than did patients in previous studies who had received the same type of chemotherapy but without the gene-modified bone marrow stem cell transplant. The authors found that chemotherapy increased the number of gene-modified blood and bone marrow cells in these patients. These patients survived longer than predicted without any negative side effects from the transplanted cells or the treatment given. This strategy could be used for treating other types of cancer, or diseases treated with the same type of chemotherapy, to increase the efficacy of the drug regimen. This strategy could also be further developed as a clinical application in other diseases where defective bone marrow stem cells can be corrected by gene therapy but need to be increased to higher levels to produce a therapeutic benefit. Chemotherapy with alkylating agents for treating malignant disease results in myelosuppression that can significantly limit dose escalation and potential clinical efficacy. Gene therapy using mutant methylguanine methyltransferase (P140K) gene–modified hematopoietic stem and progenitor cells may circumvent this problem by abrogating the toxic effects of chemotherapy on hematopoietic cells. However, this approach has not been evaluated clinically. Here, we show efficient polyclonal engraftment of autologous P140K-modified hematopoietic stem and progenitor cells in three patients with glioblastoma. Increases in P140K-modified cells after transplant indicate selection of gene-modified hematopoietic repopulating cells. Longitudinal retroviral integration site (RIS) analysis identified more than 12,000 unique RISs in the three glioblastoma patients, with multiple clones present in the peripheral blood of each patient throughout multiple chemotherapy cycles. To assess safety, we monitored RIS distribution over the course of chemotherapy treatments. Two patients exhibited emergence of prominent clones harboring RISs associated with the intronic coding region of PRDM16 (PR domain–containing 16) or the 3′ untranslated region of HMGA2 (high-mobility group A2) genes with no adverse clinical outcomes. All three patients surpassed the median survival for glioblastoma patients with poor prognosis, with one patient alive and progression-free more than 2 years after diagnosis. Thus, transplanted P140K-expressing hematopoietic stem and progenitor cells are chemoprotective, potentially maximizing the drug dose that can be administered.

Chromatin maps, histone modifications and leukemia

Recent years have seen great advances in the understanding of epigenetic gene regulation. Many of the molecular players involved have recently been identified and are rapidly being characterized in detail. Genome scale studies, using chromatin immunoprecipitation followed by expression arrays (‘ChIP-Chip’) or next generation sequencing (‘ChIP-Seq’), have been applied to the study of transcription factor binding, DNA methylation, alternative histone use, and covalent histone modifications such as acetylation, ubiquitination and methylation. Initial studies focused on yeast, and embryonic stem cells. Genome-wide studies are now also being employed to characterize cancer and specifically leukemia genomes, with the prospect of improved diagnostic accuracy and discovery of novel therapeutic strategies. Here, we review some of the epigenetic modifications and their relevance for leukemia.

Recent progress toward epigenetic therapies: the example of mixed lineage leukemia

The importance of epigenetic gene regulatory mechanisms in normal and cancer development is increasingly evident. Genome-wide analyses have revealed the mutation, deletion, and dysregulated expression of chromatin-modifying enzymes in a number of cancers, including hematologic malignancies. Genome-wide studies of DNA methylation and histone modifications are beginning to reveal the landscape of cancer-specific chromatin patterns. In parallel, recent genetic loss-of-function studies in murine models are demonstrating functional involvement of chromatin-modifying enzymes in malignant cell proliferation and self-renewal. Paradoxically, the same chromatin modifiers can, depending on cancer type, be either hyperactive or inactivated. Increasingly, cross talk between epigenetic pathways is being identified. Leukemias carrying MLL rearrangements are quintessential cancers driven by dysregulated epigenetic mechanisms in which fusion proteins containing N-terminal sequences of MLL require few or perhaps no additional mutations to cause human leukemia. Here, we review how recent progress in the field of epigenetics opens potential mechanism-based therapeutic avenues.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

Ezh2 Controls an Early Hematopoietic Program and Growth and Survival Signaling in Early T Cell Precursor Acute Lymphoblastic Leukemia

SUMMARY Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.