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PDF file - 106K, Immunoblot analysis of the indicated proteins in U-266 and LP-1 pre-treated with 10 muM CQ, 10 muM zVAD.fmk or 4 muM MG132 (for 4h) followed by 10 mu M Sor for 24h

PDF file - 26K, The indicated myeloma cell lines were treated with 10 muM Sor for 24h and cell cycle distribution was analysed by NucleoCounter NC-3000 (Chemometec).

PDF file - 36K, A, Quantitative analysis of Annexin V/PI positive murine 5T33MMvitro cells treated the indicated concentrations of sorafenib for 24h and 48h; B, Immunoblot analysis of the indicated proteins from the 5T33MMvitro cell line treated with 10 muM Sor for the indicated time points. C, Immunoblot analysis of the indicated proteins from murine 5T33MMvivo treated ex-vivo with 10 muM Sor for 24h

Recent evidence has accumulated that the dynamic histone methylation mediated by histone methyltransferases and demethylases plays key roles in regulation of chromatin structure and transcription. In the present study, we show that SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase implicated in oncogenesis, directly trans-activates the telomerase reverse transcriptase (hTERT) gene that is essential for cellular immortalization and transformation. SMYD3 occupies its binding motifs on the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation, thereby contributing to inducible and constitutive hTERT expression in normal and malignant human cells. Knocking down SMYD3 in tumor cells abolished trimethylation of H3-K4, attenuated the occupancy by the trans-activators c-MYC and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factor binding to the hTERT promoter. The present findings provide significant insights into regulatory mechanisms of hTERT/telomerase expression; moreover, identification of the hTERT gene as a direct target of SMYD3 contributes to a better understanding of SMYD3-mediated cellular transformation. [Cancer Res 2007;67(6):2626–31]

Activation of telomerase, a key event during immortalization and malignant transformation, requires expression of the telomerase reverse transcriptase (hTERT). Consistently, lack of telomerase activity and hTERT expression occurs in most normal human somatic cells. However, it has been observed that both normal and cancerous renal tissues express hTERT whereas only the latter exhibits telomerase activity. The mechanism underlying the dissociation between hTERT expression and telomerase activity is unclear. In the present study, we examined telomerase activity and alternative splicing of hTERT transcripts in renal cell carcinoma (RCC) specimens and adjacent normal tissues from 33 patients with RCC. Telomerase activity was detectable in 27 of 33 (82%) RCC samples but none in their normal counterparts. Thirty-two of 33 tumors expressed overall hTERT mRNA and 27 of them contained full-length hTERT transcripts, all with telomerase activity. Although 42% (14 of 33) of normal renal samples expressed hTERT mRNA, none of them had full-length hTERT transcripts, coinciding with lack of telomerase activity. The presence of full-length hTERT mRNA and telomerase activity was significantly associated with c-MYC induction. In tumors, absence of full-length hTERT mRNA or telomerase activity defines a subgroup of nonmetastatic, early-stage RCCs. Taken together, telomerase repression in normal renal tissues is attributed to the absence of full-length hTERT transcripts, whereas telomerase activation is achieved via induction of or switch to expression of full-length hTERT mRNA during the oncogenic process of kidneys, and associated with aggressive RCCs.

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy with high mortality rate. Treatment outcomes have improved since the introduction of new drugs such as the IMiD lenalidomide, but relapse rates and resistance is still a problem. The gene ABCB1 encodes the drug transporter p-glycoprotein (p-gp) which confers resistance through extrusion of drugs over the cell membrane. Lenalidomide is subject to limited metabolism and excreted mainly via the kidneys. In vitro studies have shown lenalidomide to be an ABCB1 substrate, and single nucleotide polymorphisms (SNPs) affecting gene expression, transporter function and/or activity may affect drug distribution and the subsequent outcome and risk of adverse events. However, in vivo studies of the effect of ABCB1 on lenalidomide pharmacokinetics are contradictory. Our aim was to investigate the influence of ABCB1 SNPs on lenalidomide treatment outcome and adverse events (AE). Materials & Methods: In the observational part of two connected studies, 133 Lenalidomide naïve patients at 1st relapse/refractory MM were treated with lenalidomide and dexamethasone for up to 9 cycles of 4 weeks. In the prospective 2nd part, 62 patients that had achieved at least partial response according to IMWG-criteria followed by at least two additional treatment cycles were randomized to either lenalidomide/dexamethasone or lenalidomide as a single drug. 90 patients (of which 47 was further randomized to the 2nd part) had samples available for genotyping of the ABCB1 SNPs 1199G>A (Ser400Asn, rs2229109), 1236C>T (rs1128503), 2677G>T/A (Ala893Ser, rs2032582) and 3435C>T (rs1045642) using Pyrosequencing. Correlations to overall survival, time to progression (TTP), response parameters and AE were investigated, and a p-value of 0.05 was considered significant. Results: No significant correlations to hematological AE or response rates were found, and no impact on survival for 1236C>T, 2677G>T/A or 3435C>T, neither in the whole population nor in patients randomized to the 2nd part. The results were similar also when risk (according to FISH) was considered. There was a trend towards improved TTP for patients carrying the 1199A variant; mean TTP 3.2 years (95%CI 2.3-4.1) vs 2.2 years (95%CI 1.8-2.6) for G/A and G/G, respectively (p=0.076). This trend was confirmed in the multivariable cox regression analysis; HR=0.280 (95%CI 0.74-1.054), p=0.06. The difference in TTP was significant in the non-high risk subgroup; mean TTP 4.3 years (95%CI 3.7-4.9) vs 2.3 years (95%CI 1.8-2.8), p=0.034, for G/A and G/G, respectively. Conclusion: No evidence was found for a large impact of 1236C>T, 2677G>T/A or 3435C>T on lenalidomide treatment outcome or risk of hematological AE. 1199G>A may be a potential marker of TTP in non-high risk MM but further studies in a larger cohort is needed to clarify the relationship and whether this is due to altered drug transport or efflux independent mechanisms. Citation Format: Ingrid Jakobsen Falk, Johan Lund, Henrik Gréen, Astrid Gruber, Evren Alici, Birgitta Lauri, Cecilie Blimark, Ulf-Henrik Mellqvist, Agneta Swedin, Karin Forsberg, Conny Carlsson, Mats Hardling, Lucia Ahlberg, Hareth Nahi, Kourosh Lotfi. The impact of ABCB1 single nucleotide polymorphisms on the outcome in lenalidomide treated multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2017-5030