Clare M. Adams

miR-223 regulates cell growth and targets proto-oncogenes in mycosis fungoides/cutaneous T-cell lymphoma.

The pathogenesis of the cutaneous T-cell lymphoma (CTCL), mycosis fungoides (MF) is unclear. MicroRNA (miRNA) are small non-coding RNA that target mRNA leading to reduced mRNA translation. Recently, specific miRNA were shown to be altered in CTCL. We identified significantly reduced expression of miR-223 in early stage MF skin, and the levels of miR-223 diminished further in advanced stage disease. CTCL peripheral blood mononuclear cells and cell lines also had reduced miR-223 as compared to controls. Elevated expression of miR-223 in these cell lines reduced cell growth and clonogenic potential, whereas inhibition of miR-223 increased cell numbers. Investigations into putative miR-223 targets with oncogenic function, including E2F1 and MEF2C, and the predicted miR-223 target, TOX, revealed all three are targeted by miR-223 in CTCL. E2F1, MEF2C, and TOX proteins were decreased with miR-223 overexpression, while miR-223 inhibition led to increased protein levels in CTCL. In addition, we showed the 3′-UTR of TOX mRNA was a genuine target of miR-223. Therefore, reduced levels of miR-223 in MF/CTCL lead to increased expression of E2F1, MEF2C, and TOX, which likely contribute to the development and/or progression of CTCL. Thus, miR-223 and its targets may be useful for the development of new therapeutics for MF/CTCL.

Inactivation of p53 Is Insufficient to Allow B Cells and B-Cell Lymphomas to Survive Without Dicer

Inactivation of p53, the master regulator of cellular stress and damage signals, often allows cells that should die or senesce to live. Loss of Dicer, an RNase III-like enzyme critical in microRNA biogenesis, causes embryonic lethality and activation of the p53 pathway. Several nonhematopoietic cell types that contain inactivated p53 have been shown to survive Dicer deletion, suggesting that p53 loss may protect cells from the negative consequences of Dicer deletion. However, here, we report that loss of p53 did not provide a survival advantage to B cells, as they underwent rapid apoptosis upon Dicer deletion. Moreover, a deficiency in p53 neither rescued the Dicer deletion-induced delay in Myc-driven B-cell lymphomagenesis, nor allowed a single B-cell lymphoma to develop with biallelic deletion of Dicer. A p53 deficiency did, however, restore the pre-B/B-cell phenotype and CD19 surface expression of the lymphomas that emerged in conditional Dicer knockout Eμ-myc transgenic mice. Moreover, p53 loss in transformed B cells did not confer protection from apoptosis, as Dicer deletion in established p53-null B-cell lymphomas induced apoptosis, and all of the 1,260 B-cell lymphoma clones analyzed that survived Cre-mediated Dicer deletion retained at least one allele of Dicer. Moreover, Dicer deletion in lymphomas in vivo reduced tumor burden and prolonged survival. Therefore, inactivation of p53 is insufficient to allow untransformed B cells and B-cell lymphomas to survive without Dicer, presenting a potential therapeutic opportunity for the treatment of B-cell lymphomas.

Myc Induces miRNA-Mediated Apoptosis in Response to HDAC Inhibition in Hematologic Malignancies

Alterations in the expression or function of histone deacetylases (HDAC) contribute to the development and progression of hematologic malignancies. Consequently, the development and implementation of HDAC inhibitors has proven to be therapeutically beneficial, particularly for hematologic malignancies. However, the molecular mechanisms by which HDAC inhibition (HDACi) induces tumor cell death remain unresolved. Here, we investigated the effects of HDACi in Myc-driven B-cell lymphoma and five other hematopoietic malignancies. We determined that Myc-mediated transcriptional repression of the miR-15 and let-7 families in malignant cells was relieved upon HDACi, and Myc was required for their upregulation. The miR-15 and let-7 families then targeted and downregulated the antiapoptotic genes Bcl-2 and Bcl-xL, respectively, to induce HDACi-mediated apoptosis. Notably, Myc also transcriptionally upregulated these miRNA in untransformed cells, indicating that this Myc-induced miRNA-mediated apoptotic pathway is suppressed in malignant cells, but becomes reactivated upon HDACi. Taken together, our results reveal a previously unknown mechanism by which Myc induces apoptosis independent of the p53 pathway and as a response to HDACi in malignant hematopoietic cells.

Histone deacetylase inhibition reveals a tumor-suppressive function of MYC-regulated miRNA in breast and lung carcinoma.

Histone deacetylase (HDAC) inhibition leads to dynamic changes in the epigenetic landscape that is postulated to alter the expression of critical mediators of cellular proliferation and death. While current HDAC inhibitors have shown to be efficacious in the treatment of specific hematologic malignancies, their therapeutic utility in epithelial-based cancers warrants further evaluation. Moreover, the mechanisms of HDAC inhibition-induced cancer cell death are not completely understood. Therefore, elucidation of the underlying pathways engaged by HDAC inhibition may enable the development of more effective therapeutic strategies. Here, we report that HDAC inhibition in human breast and lung carcinoma cells activates an apoptotic mechanism mediated by microRNA (miRNA) and induced by the oncogene MYC. Specifically, following HDAC inhibition, MYC, which normally represses miR-15 and let-7 families, transcriptionally activated their expression and MYC was required for this miRNA upregulation. As a result, transcript levels of the tumor-suppressive miR-15 and let-7 families increased, which targeted and decreased the expression of the crucial prosurvival genes BCL-2 and BCL-XL, respectively. MYC was also required for the downregulation of BCL-2 and BCL-XL following HDAC inhibition. Blocking the binding sites of the miR-15 and let-7 families in the 3′-untranslated regions of BCL-2 and BCL-XL protected against HDAC inhibition-induced apoptosis. These results provide important insight into the molecular underpinnings of HDAC inhibition-induced cell death in breast and lung cancer and reveal a tumor-suppressive role for MYC-regulated miRNA that is activated with HDAC inhibition.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitts Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

BCL-W has a fundamental role in B cell survival and lymphomagenesis

Compromised apoptotic signaling is a prerequisite for tumorigenesis. The design of effective therapies for cancer treatment depends on a comprehensive understanding of the mechanisms that govern cell survival. The antiapoptotic proteins of the BCL-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Unlike BCL-2 and BCL-XL, the closest antiapoptotic relative BCL-W is required for spermatogenesis, but was considered dispensable for all other cell types. Here, however, we have exposed a critical role for BCL-W in B cell survival and lymphomagenesis. Loss of Bcl-w conferred sensitivity to growth factor deprivation–induced B cell apoptosis. Moreover, Bcl-w loss profoundly delayed MYC-mediated B cell lymphoma development due to increased MYC-induced B cell apoptosis. We also determined that MYC regulates BCL-W expression through its transcriptional regulation of specific miR. BCL-W expression was highly selected for in patient samples of Burkitt lymphoma (BL), with 88.5% expressing BCL-W. BCL-W knockdown in BL cell lines induced apoptosis, and its overexpression conferred resistance to BCL-2 family–targeting BH3 mimetics. Additionally, BCL-W was overexpressed in diffuse large B cell lymphoma and correlated with decreased patient survival. Collectively, our results reveal that BCL-W profoundly contributes to B cell lymphoma, and its expression could serve as a biomarker for diagnosis and aid in the development of better targeted therapies.

Cell survival is dicey without Dicer

The cellular requirements for Dicer, an essential miRNA processing enzyme, and the consequences of altered levels of its expression on tumorigenesis are incompletely understood. We review the effects of Dicer loss in cells of different origin and whether loss of p53 permits cell survival and growth in the absence of Dicer.

Non-Hodgkin and Hodgkin Lymphomas Select for Overexpression of BCLW

Purpose: B-cell lymphomas must acquire resistance to apoptosis during their development. We recently discovered BCLW, an antiapoptotic BCL2 family member thought only to contribute to spermatogenesis, was overexpressed in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. To gain insight into the contribution of BCLW to B-cell lymphomas and its potential to confer resistance to BCL2 inhibitors, we investigated the expression of BCLW and the other antiapoptotic BCL2 family members in six different B-cell lymphomas. Experimental Design: We performed a large-scale gene expression analysis of datasets comprising approximately 2,300 lymphoma patient samples, including non-Hodgkin and Hodgkin lymphomas as well as indolent and aggressive lymphomas. Data were validated experimentally with qRT-PCR and IHC. Results: We report BCLW is significantly overexpressed in aggressive and indolent lymphomas, including DLBCL, Burkitt, follicular, mantle cell, marginal zone, and Hodgkin lymphomas. Notably, BCLW was preferentially overexpressed over that of BCL2 and negatively correlated with BCL2 in specific lymphomas. Unexpectedly, BCLW was overexpressed as frequently as BCL2 in follicular lymphoma. Evaluation of all five antiapoptotic BCL2 family members in six types of B-cell lymphoma revealed that BCL2, BCLW, and BCLX were consistently overexpressed, whereas MCL1 and A1 were not. In addition, individual lymphomas frequently overexpressed more than one antiapoptotic BCL2 family member. Conclusions: Our comprehensive analysis indicates B-cell lymphomas commonly select for BCLW overexpression in combination with or instead of other antiapoptotic BCL2 family members. Our results suggest BCLW may be equally as important in lymphomagenesis as BCL2 and that targeting BCLW in lymphomas should be considered. Clin Cancer Res; 23(22); 7119–29. ©2017 AACR.

Mdm2 is required for survival and growth of p53-deficient cancer cells

p53 deletion prevents the embryonic lethality of normal tissues lacking Mdm2, suggesting that cells can survive without Mdm2 if p53 is also absent. Here we report evidence challenging this view, with implications for therapeutically targeting Mdm2. Deletion of Mdm2 in T-cell lymphomas or sarcomas lacking p53 induced apoptosis and G2 cell-cycle arrest, prolonging survival of mice with these tumors. p53-/- fibroblasts showed similar results, indicating that the effects of Mdm2 loss extend to premalignant cells. Mdm2 deletion in p53-/- cells upregulated p53 transcriptional target genes that induce apoptosis and cell-cycle arrest. Mdm2 deletion also increased levels of p73, a p53 family member. RNAi-mediated attenuation of p73 rescued the transcriptional and biological effects of Mdm2 loss, indicating that p73 mediates the consequences of Mdm2 deletion. In addition, Mdm2 deletion differed from blocking Mdm2 interaction with p53 family members, as Nutlin-3 induced G1 arrest but did not activate apoptosis in p53-/- sarcoma cells. Our results indicate that, in contrast to current dogma, Mdm2 expression is required for cell survival even in the absence of p53. Moreover, our results suggest that p73 compensates for loss of p53 and that targeting Mdm2 in p53-deficient cancers has therapeutic potential. Cancer Res; 77(14); 3823-33. ©2017 AACR.

Molecular underpinnings of HDAC inhibition revealed

Aberrant expression and/or activity of chromatin modifying enzymes, such as histone deacetylases (HDACs) have been implicated in many human cancers. Consequently, HDAC inhibitors (HDACi) are being developed and tested as cancer therapeutics for multiple malignancies, and have shown efficacy in treating T-cell lymphomas. While HDAC inhibition of tumor cells can induce apoptosis and affect cellular processes, including cell cycle and DNA damage/repair, typically resulting in cell death, the underlying mechanism(s) for how this occurs has remained unclear. Understanding the cellular mechanisms causal to the anticancer activity of HDACi should lead to more efficacious HDAC inhibitors to treat human malignancies, including epithelial-based cancers, such as breast and lung adenocarcinoma. We recently revealed a previously unknown mechanism of HDACi-mediated apoptosis of human breast and lung carcinomas, which we also determined is shared in haematopoietic malignancies. Studies evaluating mRNA levels reported changes in gene expression following HDACi. However, although histone acetylation is linked to transcriptional activation, surprisingly, HDACi did not cause global transcriptional dysregulation, but did alter the mRNA levels of several BCL-2 family members. Our data from precision global run-on transcription coupled with massively paralleled sequencing (PRO-seq) showed little difference in the transcription of anti-apoptotic BCL-2 genes after HDACi. Consequently, we hypothesized post-transcriptional mechanisms, namely microRNA (miRNA), were likely involved in mediating the gene expression changes following HDACi. Breast and lung cancers, among others, often present with decreased levels of miRNA, which can be attributed to repression by transcription factors, such as MYC. Using multiple pan-class I and selective class I HDACi, we revealed a mechanism whereby MYC represses the tumor suppressor miR-15 and let-7 miRNA families with the help of HDACs to close chromatin and shut down transcription at the miRNA promoters (Fig. 1). Without MYC present, HDACi had minimal, if any, effect on the expression of the miR-15 and let-7 families. In all cell types analyzed (haematopoietic and non-haematopoietic), HDAC3 appeared to be the predominant HDAC involved in their transcriptional repression. Similarly, utilizing the same HDAC3-selective compound we evaluated and concurrent with our studies on haematological malignancies, myeloid and lymphoid malignancies were reported to be more sensitive to loss of HDAC3 than inhibition of the other class I HDACs. Therefore, HDAC3 appears to be primarily responsible for the repression of the miR-15 and let-7 families and the ensuing apoptosis. Importantly, we determined that HDACi in tumor cells altered the transcriptional program of MYC, switching MYC from a transcriptional repressor to a transcriptional activator of the miR-15 and let-7 families to increase the levels of these tumor suppressor miRNA in breast and lung cancer cells (Fig. 1). Our results indicate that more focused efforts on HDAC3 should improve the efficacy of HDACi therapies for breast and lung carcinoma. Although the miR-15 and let-7 families were transcriptionally repressed by MYC in cancer cells and re-activated by HDACi, we unexpectedly discovered that their expression was induced by dysregulated MYC in normal, non-transformed cells (Fig. 1). Given that the miR-15 and let-7 families can promote apoptosis by targeting the mRNA of pro-survival genes BCL-2 and BCL-XL, respectively, 5 we investigated whether a connection between MYC, these miRNA, and BCL-2 and BCLXL existed. Our data exposed an unknown link whereby MYC transcriptionally activates the miR-15 and let-7 families in normal cells that directly target and suppress the expression of anti-apoptotic BCL-2 and BCL-XL as a novel tumor suppressive mechanism to counter the hyper-proliferative signals of dysregulated MYC (Fig. 1). Previously, we reported that MYC-mediated apoptosis occurs, in part, by down-regulating BCL-2 and BCL-XL through a mechanism that remained incompletely understood. Our recent studies have revealed that the decreased expression of BCL-2 and BCL-XL is due to MYC transcriptionally up-regulating the miR-15 and let-7 families that target BCL-2 and BCL-XL, respectively. 3,4 Moreover, our results demonstrate that cellular transformation status dictates whether MYC transcriptionally activates or represses the miR15 and let-7 families (Fig. 1). We determined that this previously unknown miRNA-mediated mechanism of MYCinduced apoptosis in normal cells is inactivated in breast and