Cristina Cristofoletti

Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-kappaB pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS.

Comprehensive analysis of PTEN status in Sezary syndrome.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma characterized by recurrent chromosomal alterations, among which, chromosome 10q deletion is very frequent. In this study, we investigated the PTEN status, on locus 10q23, in 44 SS patients; our findings show that PTEN is deleted in 36% of SS cases, whereas PTEN downregulation is observed in almost all of the samples evaluated by quantitative reverse-transcriptase polymerase chain reaction and Western blotting analysis. Neither DNA sequence mutation nor promoter hypermethylation were found at the PTEN locus, but we demonstrate that PTEN level can be also reduced by a group of miRs previously found upregulated and of prognostic relevance in SS; particularly, miR-21, miR-106b, and miR-486 were able to control PTEN abundance either in vitro or in vivo. Finally, because reduced PTEN activates the PI3/AKT-mediated pathway of cell growth and survival, we demonstrate that PTEN deficiency is associated with activated AKT in skin resident but not circulating SS cells, suggesting that the cutaneous milieu may strongly contribute to the SS cell growth. To our knowledge, this is the first study fully exploring the PTEN status in a large cohort of SS patients, unveiling potential elements of clinical utility in this malignancy.

Clonal expansion and functional exhaustion of monoclonal marginal zone B cells in mixed cryoglobulinemia: the yin and yang of HCV-driven lymphoproliferation and autoimmunity.

Monoclonal marginal zone (MZ) B cells expressing a V(H)1-69-encoded idiotype accumulate in HCV-associated mixed cryoglobulinemia (MC). These cells recognize the E2 protein of HCV and their massive clonal expansion reflects the propensity of MZ B cells to proliferate robustly upon antigenic stimulation by microorganisms, a property that makes them prone to neoplastic transformation. V(H)1-69(+) B cells of MC patients are phenotypically heterogeneous and resemble either mature MZ B cells (IgM(+)CD27(+)CD21(high)) or the unusual CD21(low) B cells that accumulate in other immunological disorders such as common variable immunodeficiency (CVID) or HIV infection. The CD21(low) V(H)1-69(+) B cells of MC patients, like those of CVID and HIV patients, are anergic to BCR and TLR9 stimulation and display deregulation of several anergy-related genes; proliferative anergy is also observed in CD21(high) MZ-like V(H)1-69(+) B cells, that over-express the antiproliferative transcriptional repressor Stra13. Upon evolution to splenic marginal zone lymphoma, MZ-like V(H)1-69(+) B cells down-regulate Stra13 and partially recover their capacity to proliferate in response to TLR9 ligation. Like yin and yang, robust clonal expansion and early proliferative anergy may be viewed as the opposite forces balancing the responses of human MZ B cells to chronic microbial stimuli. Disruption of this balance facilitates autoimmunity and lymphoproliferation.

Clonal B cells of HCV-associated mixed cryoglobulinemia patients contain exhausted marginal zone-like and CD21 low cells overexpressing Stra13

A clonal population of B cells expressing a VH1‐69‐encoded idiotype accumulates in hepatitis C virus (HCV) associated mixed cryoglobulinemia (MC). These cells are phenotypically heterogeneous, resembling either typical marginal zone (MZ) B cells (IgM+IgD+CD27+CD21+) or the exhausted CD21low B cells that accumulate in HIV infection or in common variable immunodeficiency. We show that both the MZ‐like and the CD21low VH1‐69+ B cells of MC patients are functionally exhausted, since they fail to respond to TLR and BCR ligands. The proliferative defect of VH1‐69+ B cells can be overcome by co‐stimulation of TLR9 and BCR in the presence of interleukin(IL)‐2 and IL‐10. The MZ‐like VH1‐69+ B cells do not express the inhibitory receptors distinctive of CD21low B cells, but display constitutive activation of extracellular signal regulated kinase (ERK) and attenuated BCR/ERK signaling. These cells also express abundant transcripts of Stra13 (DEC1, Bhlhb2, Sharp2, Clast5), a basic helix‐loop‐helix transcription factor that acts as a powerful negative regulator of B‐cell proliferation and homeostasis. Our findings suggest that MZ B cells activated by HCV undergo functional exhaustion associated with BCR signaling defects and overexpression of a key antiproliferative gene, and may subsequently become terminally spent CD21low B cells. Premature exhaustion may serve to prevent the outgrowth of chronically stimulated MZ B cells.

T Cell Leukemia/Lymphoma 1A is essential for mouse epidermal keratinocytes proliferation promoted by insulin-like growth factor 1

T Cell Leukemia/Lymphoma 1A is expressed during B-cell differentiation and, when over-expressed, acts as an oncogene in mouse (Tcl1a) and human (TCL1A) B-cell chronic lymphocytic leukemia (B-CLL) and T-cell prolymphocytic leukemia (T-PLL). Furthermore, in the murine system Tcl1a is expressed in the ovary, testis and in pre-implantation embryos, where it plays an important role in blastomere proliferation and in embryonic stem cell (ESC) proliferation and self-renewal. We have also observed that Tcl1-/- adult mice exhibit alopecia and deep ulcerations. This finding has led us to investigate the role of TCL1 in mouse skin and hair follicles. We have found that TCL1 is expressed in the proliferative structure (i.e. the secondary hair germ) and in the stem cell niche (i.e. the bulge) of the hair follicle during regeneration phase and it is constitutively expressed in the basal layer of epidermis where it is required for the correct proliferative–differentiation program of the keratinocytes (KCs). Taking advantage of the murine models we have generated, including the Tcl1-/- and the K14-TCL1 transgenic mouse, we have analysed the function of TCL1 in mouse KCs and the molecular pathways involved. We provide evidence that in the epidermal compartment TCL1 has a role in the regulation of KC proliferation, differentiation, and apoptosis. In particular, the colony-forming efficiency (CFE) and the insulin-like growth factor 1 (IGF1)-induced proliferation are dramatically impaired, while apoptosis is increased, in KCs from Tcl1-/- mice when compared to WT. Moreover, the expression of differentiation markers such as cytokeratin 6 (KRT6), filaggrin (FLG) and involucrin (IVL) are profoundly altered in mutant mice (Tcl1-/-). Importantly, by over-expressing TCL1A in basal KCs of the K14-TCL1 transgenic mouse model, we observed a significant rescue of cell proliferation, differentiation and apoptosis of the mutant phenotype. Finally, we found TCL1 to act, at least in part, via increasing phospho-ERK1/2 and decreasing phospho-P38 MAPK. Hence, our data demonstrate that regulated levels of Tcl1a are necessary for the correct proliferation and differentiation of the interfollicular KCs.

DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect.

Abstract 936: Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome

Introduction Sezary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients. Methods Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement. Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines. Results Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P = 0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc = 4.83), mTOR (Fc = 4.61) mP70S6K (Fc = 4.64) BAD (Fc = 7.09) and 4EBP1 (Fc = 5.40)in skin-SS cells respect to circulating ones. We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients.. Conclusion: Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome. Citation Format: Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 936.

Abstract 4178: Tcl1 enhances keratinocytes’ survival/proliferation by promoting erk and jnk/sap phosphorylation at the expense of p38 and by controlling c-fos expression through miR-29b and miR-181a-1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Tcl1 oncogene has been initially isolated for its involvement in chromosomal translocations of T-prolymphocytic leukemias and overexpression in B chronic lymphocytic leukemia by enhancing AKT nuclear translocation and allowing the Ser-473 transphosphorylation. Tcl1, also, acts as AP-1 transcriptional inhibitor, by interacting with c-fos and c-jun. More recent works have associated Tcl1 to proliferation and self-renewal ability of ES cells under the direct activation of OCT3/4, Zfx, KLF5 transcription factors, being Tcl1 expressed in preimplantation embryos where it allows the progression beyond the 4-cells stage. We have recently shown that the Tcl1 oncogene is expressed in epidermis and defines secondary hair germ (transient-amplifying, TA) cells differentiation at catagen-telogen (the degenerative-resting phase of the hair follicle (HF)) transition, allowing the proliferation of TA cells in anagen (regenerative phase of the HF), giving the slow-cycling stem cells, the ability to incorporate BrdU. In fact, Tcl1 mutant (Tcl1-/-) affects stem-cell marker CD34 expression and BrdU incorporation in the bulge and TA cells, resulting in skin defects in adults with the onset of alopecia followed by skin wounding. Phenomena that are almost completely rescued by K14-TCL1 transgenic expression, in vivo. Since Tcl1 has a role in maintenance of a normal skin homeostasis in mice, involving both hair growth and epidermis, we used the approach of the expression chip analysis to unravel the pathways that are affected by loss of function and overexpression of Tcl1 in epidermal keratinocytes, by using Tcl1-/- and K14-TCL1;Tcl1-/- mice models. Our findings show that Tcl1 function involves the MAPK pathway, since Tcl1-/- shows increasing in p38MAPK phosphorylation linked to terminal differentiation/senescence/apoptosis of keratinocytes, while K14-driven overexpression shows increasing of p-Erk and p-Sapk/p-Jnk phosphorylations, linked to proliferation/commitment of keratinocytes. These signals flow through the MAPK cascade lead to altered AP1 factor function. In particular, the phosphorylation of AP-1 subunit c-Jun and c-fos transcriptional regulation and cellular localization result also affected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4178. doi:1538-7445.AM2012-4178

Abstract 150: Regulation of TGFB receptor by miR21 in Sezary syndrome

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Sezary syndrome (SS) represents a very aggressive form of Cutaneous T Cell Lymphomas with a median overall survival of 5.1 years (range, 0.4-18.6 years). As it appears evident there are in this disease either short survivors than long survivors. Previous studies of prognostic indicators in SS showed that circulating Sezary cell count, high CD4/CD8 ratio, advanced age, high lactate dehydrogenase serum level and a high white blood cell count were associated with an unfavorable outcome. Up to now, few data have been provided concerning possible associations between immunological and genetic markers, who might provide also clues on tumor progression in this disease. In the last 10 years we have observed more than 50 Sezary Syndromes in our Institute and phenotyped them with new immunologic markers. We have also performed genetic analysis with Single Nucleotide polymorphysms (SNPs) for evaluation of genomic imbalances, mRNA expression and lately microRNA(miRNA) expression profiling. In this study we have analyzed the expression profile of 470 miRNAs using Agilent platform array in 22 SS patients. We investigated the relationship between the expression level of miRNAs and the clinical outcome of SS patients by Kaplan-Meier method and risk assessment by multivariate analysis. We also functionally investigated the role of miR-21, mapping in one of the region more frequently amplified in SS and some of its targets. We identified 45 miRNAs differentially expressed between SS and healthy controls. Using predictive analysis, a list of 19 miRNAs, including miR-21 and miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs able to discriminate patients with unfavorable and favorable outcome. We show that miR-21 knockdown increases apoptosis and modulates TGF beta receptor 2 expression in vitro. The antipoptotic effect appears to be regulated through PTEN. Conversely, we were not able to observe a direct miR-21 regulation on PDCD4 gene mapping to chromosome 10q24, a frequently imbalanced region in SS. In conclusion we have identified a new prognostic miRNAs signature in SS and characterized the role of miR-21, one of the most involved in cancer, recognized as a disease and prognostic classifier in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 150. doi:10.1158/1538-7445.AM2011-150