Page 229 - Haematologica May 2020

Genomic alterations in high-risk CLL
Figure 7. Disruption of the NOTCH1 transcription repressor complex adds to the overall frequency of altered NOTCH1 signaling in high-risk chronic lymphocytic leukemia. (A) Minimal consensus region of del(4)(p15) covering the RBPJ gene locus. Raw log2 ratio, chromosome 4, cases #2O_CLL036 and #2O_CLL068 displayed with the UCSC genome browser (hg18). Red bars represent determined log2 ratios of single probe sets sorted by their physical position along the chromosome. The minimal consensus region of del(4)(p15) harboring the RBPJ gene locus is shown. (B) RBPJ expression levels in cases with del(4)(p15). RBPJ gene expression levels were calculated relative to ACTB expression levels and fold changes (FC) were calculated towards the median DCt value of all reference samples (Ref). Median expression levels within each group of samples are highlighted and the difference between the two groups was analyzed by the Mann-Whitney test. Only 4p-deleted cases with a log2 ratio lower than 0.89 were included. (C) Minimal consensus region of del(14)(q24.3) covering the SNW1 gene locus. Raw log2 ratio, chromosome 14, case #2O_CLL027. The minimal consensus region of del(14)(q24.3) harboring the SNW1 gene locus is shown. (D) SNW1 expression levels in cases with del(14)(q24.3). SNW1 gene expression levels were calculated relative to ACTB expression levels and FC were calculated towards the median DCt value of all refer- ence samples (Ref). Median expression levels within each group of samples are highlighted and the difference between the two groups was analyzed by the Mann- Whitney test. Only 14q-deleted cases with a log2 ratio lower than 0.87 were included. (E) Composition of the NOTCH1 transcription repressor complex. Simplified illustration of the NOTCH1 transcription repressor complex. The mutation frequency of SPEN is based on targeted next-generation sequencing results on 108 cases from the high-risk cohort. The deletion frequencies of RBPJ and SNW1 are based on the entire high-risk cohort (n=146). SNW1/SKIP is an unconfirmed component of the NOTCH1 repressor complex, which has been associated with the recruitment of histone deacetylases. (F) HES1, DTX1 and MYC expression levels in cases with genomic alterations affecting the NOTCH1 transcription repressor complex. HES1, DTX1 and MYC gene expression levels were calculated relative to 18S expres- sion levels and FC were calculated towards the median DCt value of all reference samples (Ref). Median expression levels within each group of samples are high- lighted and differences between groups were analyzed by the Mann-Whitney tests. Non-purified peripheral blood mononuclear cells with a tumor cell load >70% were used for the experiment. Reference samples without evidence of a genetic alteration affecting NOTCH1 signaling were taken from the CLL8 trial (favorable risk cases with 13q deletion as sole abnormality in routine fluorescence in situ hybridization analysis; light green) and from the CLL2O trial (high risk cases; dark green). Additionally, three cases with a highly clonal NOTCH1 mutation were included (blue). Cases with RBPJ alteration are shown in dark red, samples with SNW1 alteration in orange, and samples with SPEN alteration in pale red. Samples with additional NOTCH1 mutation are indicated in blue within their respective sample group. The DTX1 gene locus is located on chromosome 12. In the figure illustrating DTX1 expression levels, cases without trisomy 12 are indicated by a round symbol and cases with trisomy 12 are indicated by a square symbol. Trisomy 12 appeared to be enriched in samples with mutations affecting the repressor complex. A gene dosage effect on DTX1 gene expression was not apparent.
MYC, which comprises transcriptional inhibitors such as MGA,42 is poorly understood. The CDKN2A/B gene loci encode cell-cycle regulators decelerating cell proliferation at multiple levels.43 The CDKN2A gene product p14ARF tightly controls pro-proliferative activities of MYC so that reduced CDKN2A expression in combination with increased MYC expression can result in accelerated prolif- eration.44 Enrichment of CDKN2A/B loss and MYC gain in the relapsedTP53-/refractory cohort and frequent co- occurrence of both aberrations therefore hints at a key role for deficient cell-cycle control in the development of high-risk CLL.
The small focal gains within the 8q24.21 super- enhancer region are relevant with regard to them harbor- ing binding sites for the NICD1 transcription factor.38 MYC is a well established target gene of NOTCH1,39 and tight bonds between aberrantly strong NOTCH1 signal- ing and increased MYC activity have been observed in T- cell acute lymphoblastic leukemia and were also shown to exist in CLL.38,39 Hence, genomic aberrations with an activating effect on NOTCH1 signaling strength can indi- rectly increase the number of cases with enhanced MYC activity. Besides coding activating NOTCH1 mutations, which prolong NICD1 transcription factor activity,45 vari- ous other genomic alterations were identified to interfere with NOTCH1 signaling. These alterations include non- coding NOTCH1 mutations in the 3’ untranslated region,46 FBXW7 mutations,47 MED12 mutations,48 as well as SPEN mutations and probably also RBPJ alterations.
The fact that NOTCH1 and SF3B1 mutations are often mutually exclusive raises the question as to what extent SF3B1 mutation can disturb the physiological balance between NOTCH1 signaling and MYC transcription. This question is based on the observation that the strength of NOTCH1 signaling depends on DVL2, which inhibits transcriptional activation by NOTCH1.49 Mutations in SF3B1 lead to alternative splicing of DVL2
and the resulting splice variant has been shown to lack its ability to modulate NOTCH1 signaling.50 SF3B1 muta- tions may, therefore, constitute another frequent mecha- nism to strengthen the NOTCH1-MYC signaling axis.
Taken together, our results raise the hypothesis that mul- tiple genetic lesions in high risk CLL converge in upregulat- ed MYC activity. Testing this hypothesis requires an inte- grative analysis of the genome, transcriptome and pro- teome in samples strictly processed at 4°C and in the absence of calcium chelators. For translation of our results into clinical practice, a systematic record of genomic alter- ations identified as meaningful in our study needs to be obtained in more recent, prospective clinical trials includ- ing treatment arms based on BTK or Pi3K inhibition and/or antagonism of BCL-2. Novel potential markers must be tested for relevance in each treatment arm and markers proving to be relevant must be utilized for the assembly of a genomic clinical database. In the long-term, such an approach will allow an estimation of the likelihood of ben- efit or disadvantage from a given treatment regimen, hence paving the way towards more personalized treatment choices in the future management of CLL patients.
Acknowledgments
The authors would like to thank the patients who participated in the CLL2O, CLL8, and CLL11 trials; the investigators who treated patients and submitted samples; and Christina Galler, Sabrina Rau, and Jacqueline Fiegel for excellent assistance and support.
Funding
This study was supported by the German Research Foundation (DFG: ED 256/1 1; SFB 1074/B2 and Z1), by grants from the German Federal Ministry of Education and Research (BMBF, PRECiSe) and by the Barts Charity Fund. Central genetic diagnostics were partly funded by F. Hoffmann- La Roche.
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