Unlocking adult T-cell leukemia/lymphoma's epigenetic secrets: delving into the mechanism and impact of EZH1/2 inhibition.

Epigenetic modifications, particularly through methylation of DNA packaging histones, play a pivotal role in controlling gene expression. Aberrant patterns of histone methylation have been associated with the development and progression of hematological malignancies. Unraveling the impact of aberrant histone marks on gene expression and leukemogenesis has spurred a concerted effort to develop clinically effective epigenetic therapies. In malignancies associated with the accumulation of histone H3 lysine trimethylation (H3K27me3), one such intervention involves preventing the deposition of this repressive histone mark by inhibiting the histone-modifying enzymes EZH1 and EZH2. While inhibition of EZH1/2 has demonstrated efficacy in both preclinical studies and clinical trials in various cancers, studies delineating the dynamic effect of EZH1/2 inhibition on H3K27me3 and disease relapse in clinical samples are lacking. In a recent publication, Yamagishi et al. explore how responses of a patient with adult T-cell leukemia/lymphoma to valemetostat, an EZH1/2 inhibitor, are associated with changes in H3K27me3, chromatin accessibility and gene expression, and how these changes can be circumvented in relapsed disease.

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.

Cre recombinase expression cooperates with homozygous FLT3 internal tandem duplication knockin mouse model to induce acute myeloid leukemia.

Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration. We report on a transgenic murine model of FLT3-ITD induced disease, where Cre recombinase expression alone, and in the absence of a conditional allele, gives rise to an aggressive leukemia phenotype. Here, expression of various Cre recombinases leads to polyclonal expansion of FLT3ITD/ITD progenitor cells, induction of a differentiation block and activation of Myc-dependent gene expression programs. Our report is intended to alert the scientific community of potential risks associated with using this specific mouse model and of unexpected effects of Cre expression when investigating cooperative oncogenic mutations in murine models of cancer.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.

A knockout combination for MPN stem cells.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that are maintained by stem cell populations. In this issue of JEM, Dagher et al. (https://doi.org/10.1084/jem.20201268) combine arsenic and interferon α to deliver a knockout punch to MPN stem cells and provide new hope to cure patients with MPNs.

MPN: The Molecular Drivers of Disease Initiation, Progression and Transformation and their Effect on Treatment.

Myeloproliferative neoplasms (MPNs) constitute a group of disorders identified by an overproduction of cells derived from myeloid lineage. The majority of MPNs have an identifiable driver mutation responsible for cytokine-independent proliferative signalling. The acquisition of coexisting mutations in chromatin modifiers, spliceosome complex components, DNA methylation modifiers, tumour suppressors and transcriptional regulators have been identified as major pathways for disease progression and leukemic transformation. They also confer different sensitivities to therapeutic options. This review will explore the molecular basis of MPN pathogenesis and specifically examine the impact of coexisting mutations on disease biology and therapeutic options.

Hematopoietic stem and progenitor cell-restricted Cdx2 expression induces transformation to myelodysplasia and acute leukemia.

The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.

Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity.

It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies.

Distinct effects of ruxolitinib and interferon-alpha on murine JAK2V617F myeloproliferative neoplasm hematopoietic stem cell populations.

JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population. Whereas ruxolitinib inhibits Stat5 activation in erythroid progenitor populations, it fails to inhibit this same pathway in HSCs. In contrast, IFN-α has direct effects on HSCs. Furthermore, STAT1 phosphorylation and pathway activation is greater after IFN-α stimulation in Jak2V617F murine HSCs with increased induction of reactive oxygen species, DNA damage and reduction in quiescence after chronic IFN-α treatment. Interestingly, ruxolitinib does not block IFN-α induced reactive oxygen species and DNA damage in Jak2V617F murine HSCs in vivo. This work provides a mechanistic rationale informing how pegylated IFN-α reduces JAK2V617F allelic burden in the clinical setting and may inform future clinical efforts to combine ruxolitinib with pegylated IFN-α in patients with MPN.

Changes in long-range rDNA-genomic interactions associate with altered RNA polymerase II gene programs during malignant transformation.

The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.

Determination of the physiological and pathological roles of E2F3 in adult tissues.

While genetically engineered mice have made an enormous contribution towards the elucidation of human disease, it has hitherto not been possible to tune up or down the level of expression of any endogenous gene. Here we describe compound genetically modified mice in which expression of the endogenous E2f3 gene may be either reversibly elevated or repressed in adult animals by oral administration of tetracycline. This technology is, in principle, applicable to any endogenous gene, allowing direct determination of both elevated and reduced gene expression in physiological and pathological processes. Applying this switchable technology to the key cell cycle transcription factor E2F3, we demonstrate that elevated levels of E2F3 drive ectopic proliferation in multiple tissues. By contrast, E2F3 repression has minimal impact on tissue proliferation or homeostasis in the majority of contexts due to redundancy of adult function with E2F1 and E2F2. In the absence of E2F1 and E2F2, however, repression of E2F3 elicits profound reduction of proliferation in the hematopoietic compartments that is rapidly lethal in adult animals.

Genomic characterisation of Eµ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene.

The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.

Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma.

UNLABELLED: Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eµ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. SIGNIFICANCE: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eµ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer.

Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible "exon-switch" approach to knock in the constitutively active Pik3ca(H1047R) mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca(H1047R) mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild-type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3ca(H1047R) mutant mice died earlier (median survival 46.5 d post-mutation induction) than wild-type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.

Dysregulation of the basal RNA polymerase transcription apparatus in cancer.

Mutations that directly affect transcription by RNA polymerases rank among the most central mediators of malignant transformation, but the frequency of new anticancer drugs that selectively target defective transcription apparatus entering the clinic has been limited. This is because targeting the large protein-protein and protein-DNA interfaces that control both generic and selective aspects of RNA polymerase transcription has proved extremely difficult. However, recent technological advances have led to a 'quantum leap' in our comprehension of the structure and function of the core RNA polymerase components, how they are dysregulated in a broad range of cancers and how they may be targeted for 'transcription therapy'.

AKT signalling is required for ribosomal RNA synthesis and progression of Eµ-Myc B-cell lymphoma in vivo.

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eµ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eµ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.

The mTORC1 inhibitor everolimus prevents and treats Eµ-Myc lymphoma by restoring oncogene-induced senescence.

UNLABELLED: MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in Eµ-Myc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of Eµ-Myc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established Eµ-Myc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes. SIGNIFICANCE: This work provides novel insights into the requirements for MYC-induced oncogenesis by showing that mTORC1 activity is necessary to bypass senescence during transformation of B lymphocytes. Furthermore, tumor eradication through senescence elicited by targeted inhibition of mTORC1 identifies a previously uncharacterized mechanism responsible for significant anticancer activity of rapamycin analogues and serves as proof-of-concept that senescence can be harnessed for therapeutic benefit

Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53.

Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.