Novel Long Non Coding RNA Blackmamba Is Associated to ALK^- anaplastic Large Cell Lymphoma

Systemic Anaplastic Large Cell Lymphomas (ALCLs) is among the common subtypes of T-cell lymphomas. The prognosis of ALCLs patients has been proven to be associated with translocations of the anaplastic lymphoma kinase (ALK) gene, having ALK negative patients (ALK-ALCL) a poorer 5-years survival rate (30-40%). We now recognize that ALK- ALCL includes multiple subsets some of which are bear unique genetic defects and unique clinical outcomes. Long non coding RNAs (lncRNAs) are regulators of gene expression controlling critical processes (epigenetic regulation, imprinting, cell cycle, apoptosis) frequently deregulated in cancer. In T-cells, more than 500 lncRNAs are associated with lymphocyte signatures, control cell differentiation and cell identity. While lncRNAs are found to be differentially expressed in solid cancer and directly linked to the different stages of carcinogenesis, their role in lymphoid neoplasms is largely unknown.

To identify novel lncRNAs maintaining the neoplastic phenotype of ALCL, we prepared cDNA libraries representative of coding and non-coding RNA. High coverage and directional RNA-sequencing was performed in 30 purified human T lymphocytes from multiple healthy donors, corresponding to different stages of differentiation, 22ALK+ ALCL and 20 ALK-ALCL patients' samples. Seven bona fide ALCL cell lines were included.

By de novo transcriptome reconstruction and using a new bioinformatic pipeline, we identified 83lncRNAs exclusively expressed in ALCL patient's samples. Among them, 82 lncRNAs were coshared between ALK+ and ALK- ALCL and only one was exclusively expressed in ALK-ALCL samples. We named this new lncRNA BlackMamba.

We selected the 10 top-scoring ALCL-associated lncRNAs and BlackMamba and determined their expression by RT-PCR, in a validation set of ALCL (ALK+: n=3, ALK-: n=3), T-ALL (n=8), PTCL (n=9), and AITL (n=9) samples. Resting and activated PBMCs from two additional donors were included.

Our data confirmed that the 10 top-scoring ALCL-associated lncRNAs were expressed exclusively in ALCLs without differences in the expression among ALCL subtypes. Moreover, BlackMamba was detected only in ALCL samples with a preferential expression in ALK^-ALCL samples. All lncRNAs were not expressed in normal T-lymphocytes.

We next determine the BlackMamba expression in ALCL (n=6), T-ALL (n=3), cutaneous T-lymphomas (n=3) cell lines by RT-PCR. FedP, MAC1 and MAC2a (ALK-ALCL) cell lines showed the highest expression.

Because the cellular localization of lncRNAs affects their function, we assessed the sub-cellular localization of BlackMamba by nucleo/cytoplasm cell fractionation and RT-PCR in FedP and MAC2A cell lines. BlackMamba displayed a preferential nuclear/chromatin-associated localization. Having proven that JAK/STAT signaling pathway plays a key role in both ALK+ and ALK^-ALCL, we test whether the expression of BlackMamba could be modulated after treatment with JAK1/2 (ruxolitinib) TKi. Our data showed that, in FedP and MAC2A cell lines, BlackMamba expression was 50% down-regulated relative to control.

Next, to identify whether BlackMamba may have a unique associated to specific chromatin remodel proteins, we verified its association with several candidates and found the physical interaction with the histone-lysine N-methyltransferase enzyme EZH2 by RIP in the FedP cells.

Notably, after BlackMamba knock down, the proliferation of FedP cells was reproducibly reduced (25% at 84hrs using multiple RNAi oligonucleotides).

Collective, our data indicate that ALCL aberrantly express novel and uncharacterized lncRNAs and that BlackMamba is a novel lncRNA associated with ALK^-ALCL. Moreover, BlackMamba may contributes to the maintenance of ALK-ALCL neoplastic phenotype.