Alessandra Insinga

Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway

Histone deacetylases (HDACs) regulate transcription and specific cellular functions, such as tumor suppression by p53, and are frequently altered in cancer. Inhibitors of HDACs (HDACIs) possess antitumor activity and are well tolerated, supporting the idea that their use might develop as a specific strategy for cancer treatment. The molecular basis for their selective antitumor activity is, however, unknown. We investigated the effects of HDACIs on leukemias expressing the PML-RAR or AML1-ETO oncoproteins, known to initiate leukemogenesis through deregulation of HDACs. Here we report that: (i) HDACIs induce apoptosis of leukemic blasts, although oncogene expression is not sufficient to confer HDACI sensitivity to normal cells; (ii) apoptosis is p53 independent and depends, both in vitro and in vivo, upon activation of the death receptor pathway (TRAIL and Fas signaling pathways); (iii) TRAIL, DR5, FasL and Fas are upregulated by HDACIs in the leukemic cells, but not in normal hematopoietic progenitors. These results show that sensitivity to HDACIs in leukemias is a property of the fully transformed phenotype and depends on activation of a specific death pathway.

Links between Tumor Suppressors: p53 Is Required for TGF-β Gene Responses by Cooperating with Smads

The p53 tumor suppressor belongs to a family of proteins that sense multiple cellular inputs to regulate cell proliferation, apoptosis, and differentiation. Whether and how these functions of p53 intersect with the activity of extracellular growth factors is not understood. Here, we report that key cellular responses to TGF-beta signals rely on p53 family members. During Xenopus embryonic development, p53 promotes the activation of multiple TGF-beta target genes. Moreover, mesoderm differentiation is inhibited in p53-depleted embryos. In mammalian cells, the full transcriptional activation of the CDK inhibitor p21(WAF1) by TGF-beta requires p53. p53-deficient cells display an impaired cytostatic response to TGF-beta signals. Smad and p53 protein complexes converge on separate cis binding elements on a target promoter and synergistically activate TGF-beta induced transcription. p53 can physically interact in vivo with Smad2 in a TGF-beta-dependent fashion. The results unveil a previously unrecognized link between two primary tumor suppressor pathways in vertebrates.

Impairment of p53 acetylation, stability and function by an oncogenic transcription factor.

Mutations of p53 are remarkably rare in acute promyelocytic leukemias (APLs). Here, we demonstrate that the APL‐associated fusion proteins PML–RAR and PLZF‐RAR directly inhibit p53, allowing leukemic blasts to evade p53‐dependent cancer surveillance pathways. PML–RAR causes deacetylation and degradation of p53, resulting in repression of p53 transcriptional activity, and protection from p53‐dependent responses to genotoxic stress. These phenomena are dependent on the expression of wild‐type PML, acting as a bridge between p53 and PML–RAR. Recruitment of histone deacetylase (HDAC) to p53 and inhibition of p53 activity were abrogated by conditions that either inactivate HDACs or trigger HDAC release from the fusion protein, implicating recruitment of HDAC by PML–RAR as the mechanism underlying p53 inhibition.

Recruitment of the Histone Methyltransferase SUV39H1 and Its Role in the Oncogenic Properties of the Leukemia-Associated PML-Retinoic Acid Receptor Fusion Protein

ABSTRACT Leukemia-associated fusion proteins establish aberrant transcriptional programs, which result in the block of hematopoietic differentiation, a prominent feature of the leukemic phenotype. The dissection of the mechanisms of deregulated transcription by leukemia fusion proteins is therefore critical for the design of tailored antileukemic strategies, aimed at reestablishing the differentiation program of leukemic cells. The acute promyelocytic leukemia (APL)-associated fusion protein PML-retinoic acid receptor (RAR) behaves as an aberrant transcriptional repressor, due to its ability to induce chromatin modifications (histone deacetylation and DNA methylation) and silencing of PML-RAR target genes. Here, we indicate that the ultimate result of PML-RAR action is to impose a heterochromatin-like structure on its target genes, thereby establishing a permanent transcriptional silencing. This effect is mediated by the previously described association of PML-RAR with chromatin-modifying enzymes (histone deacetylases and DNA methyltransferases) and by recruitment of the histone methyltransferase SUV39H1, responsible for trimethylation of lysine 9 of histone H3.

DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions

DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.

The emerging role of p53 in stem cells.

Among the hundreds of oncogenes and tumor suppressors that have been identified in the past 50 years, p53 is probably the best characterized; nevertheless, new functions are constantly being discovered. As a tumor suppressor, p53 regulates cellular responses to different stress stimuli by inducing reversible cell cycle arrest and DNA repair, or triggering senescence or apoptosis. Recent findings on the regulation of stem cell (SC) division and reprogramming suggest the intriguing possibility that p53 also carries out its tumor suppression function by regulating SC homeostasis. Specifically, p53 activation may counteract SC expansion by several emerging mechanisms including restriction of self-renewing divisions, inhibition of symmetric division and block of reprogramming of somatic/progenitor cells into SCs.

Mechanisms of selective anticancer action of histone deacetylase inhibitors.

Histone deacetylases (HDACs) regulate transcription and specific functions, such as tumor suppression by p53, and are frequently altered in cancer. Inhibitors of HDACs (HDACI) possess anti-tumor activity and are well tolerated, suggesting that they might develop into a specific strategy for cancer treatment. Indeed, HDACIs have successfully entered clinical trials, but the molecular basis for their selective anti-tumor activities is not clear. Recent work on leukemias expressing the PML-RAR or AML1-ETO oncogenes, known to initiate leukemogenesis through deregulation of HDACs, shows that HDACIs induce massive blast-cell apoptosis. Interestingly, the pro-apoptotic activity of the drug is not due to the relief of oncogene-mediated inhibition of the p53 tumor-suppressor pathway but, instead, relies on the selective upregulation of the death receptors DR5 and Fas and their cognate ligands TRAIL and FasL. Significantly, normal myeloid progenitors are not sensitive to HDACI-induced apoptosis and oncogene expression is not sufficient to confer HDACI-sensitivity to normal cells, demonstrating that sensitivity to HDACI is a property of the fully transformed phenotype. In principle, our findings could thus apply to other cancers, where the contribution of HDACs to tumorigenesis is not yet defined.

DNA damage response in adult stem cells.

This review discusses the processes of DNA-damage-response and DNA-damage repair in stem and progenitor cells of several tissues. The long life-span of stem cells suggests that they may respond differently to DNA damage than their downstream progeny and, indeed, studies have begun to elucidate the unique stem cell response mechanisms to DNA damage. Because the DNA damage responses in stem cells and progenitor cells are distinctly different, stem and progenitor cells should be considered as two different entities from this point of view. Hematopoietic and mammary stem cells display a unique DNA-damage response, which involves active inhibition of apoptosis, entry into the cell-cycle, symmetric division, partial DNA repair and maintenance of self-renewal. Each of these biological events depends on the up-regulation of the cell-cycle inhibitor p21. Moreover, inhibition of apoptosis and symmetric stem cell division are the consequence of the down-regulation of the tumor suppressor p53, as a direct result of p21 up-regulation. A deeper understanding of these processes is required before these findings can be translated into human anti-aging and anti-cancer therapies. One needs to clarify and dissect the pathways that control p21 regulation in normal and cancer stem cells and define (a) how p21 blocks p53 functions in stem cells and (b) how p21 promotes DNA repair in stem cells. Is this effect dependent on p21s ability to inhibit p53? Such molecular knowledge may pave the way to methods for maintaining short-term tissue reconstitution while retaining long-term cellular and genomic integrity.

Leukemia-Associated Fusion Proteins: Multiple Mechanisms of Action to Drive Cell Transformation

Leukemic cells are defined by two main biological features: arrest ofdifferentiation at a specific stage compatible with continuedproliferation, and enhanced resistance to stress. Recent work showsthat the leukemia-associated fusion protein PML-RAR can mediate bothbiological effects targeting independent pathways, through a unifyingmechanism. Differentiation block is achieved through transcriptionalsilencing of genes physiologically regulated by RAR, which are involvedin hematopoietic differentiation. In contrast, enhanced resistance tostress is due to the capacity of the fusion protein to cause degradationof the tumor suppressor p53, thus explaining the puzzling observationthat mutations of p53 are remarkably rare in acute myeloid leukemias(AMLs). Interestingly, this latter phenomenon depends on expression ofwild-type PML, acting as a molecular bridge between p53 and the fusionprotein. Strikingly, both effects require a unifying molecular mechanism:aberrant recruitment of histone deacetylases (HDACs). Therefore, thestudy of this form of leukemia appears also of interest for a betterunderstanding of the action of HDAC inhibitors, potential antitumordrugs that are at the early stages of clinical studies.

Abstract IA33: Regulation of self-renewal in cancer stem cells.

We showed that extended self-renewal of leukemia stem-cells (LSCs) is the consequence of the constitutive activation of the cell-cycle inhibitor p21. The pool of LSCs is markedly reduced in p21-/- APLs, and the residual LSCs hyperproliferate and accumulate massive DNA-damage (DD). Notably, p21 is indispensable to maintain the pool of LSCs in PML-RAR leukaemias (PML-RAR – p21-/- leukaemias are not transplantable in syngenic mice). It is not known whether p21 extends the self-renewal of LSCs by activating DD repair, restricting the cell-cycle or regulating their survival. Preliminary results from our lab showed that p21-/- APLs re-acquire the ability to initiate leukemogenesis when transplanted into immunodeficient mice (RAG) or syngeneic mice after γ-irradiation. In these conditions, the growing leukemias are indistinguishable from primary WT APLs and can be re-transplanted into immunodeficient, but not syngeneic mice, suggesting that expansion of p21-/- LSCs is controlled by the host immune-system. Further experiments suggested that p21-/- LSCs are cleared by T-cells since p21-/- APLs: i) initiate leukemias when transplanted into RAG1-/- mice (which lack B, T and NKT cells), but not in JHT mice (lacking only B-cells), and ii) do not grow in NOD/SCID mice when the recipients are injected with a T cell population primed with p21-/- blasts (e.g. obtained from the spleen of WT mice injected with p21-/- APLs), but not with non-primed T cells or T cells primed with WT blasts. T-cells primed with p21-/- blasts are also able to suppress the in vivo growth of wt APLs, but not of T-ALLs. We hypothesize that the immune-surveillance contributes to the elimination of DNA-damaged SCs, and that DNA-damaged LSCs have developed evasion strategies based on p21 activation. Citation Format: Maria Vittoria Verga Falzacappa, Alessandra Insinga, Pier Giuseppe Pelicci. Regulation of self-renewal in cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA33.