SETD2 Loss of Function Is a Recurrent Event in Advanced-Phase Chronic Myeloid Leukemia

In recent years, deregulation of epigenetic modifiers has turned out to be a recurrent event in cancer evolution. The SETD2 tumor suppressor gene encodes a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone H3 (H3K36Me3), the major chromatin mark associated with active transcription and alternative splicing. Recent studies have also implicated SETD2 in homologous recombination repair, mismatch repair, mitosis and cytokinesis. SETD2 loss of function was initially uncovered in clear cell renal cell carcinomas and was subsequently found in many other solid tumors. More recently, it has also been reported in a variety of hematologic myeloid and lymphoid malignancies, including acute lymphoblastic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphoma, systemic mastocytosis and myeloproliferative neoplasms. We thus wondered whether advanced-phase chronic myeloid leukemia (CML), that shareS similarities with acute leukemias including a high level of genetic instability, may also exhibit SETD2 loss of function.

To this purpose, we studied a relatively large cohort of 69 advanced phase CML patients (pts), including pts with accelerated phase (AP; n=15), myeloid blast crisis (my-BC; n=30), and lymphoid blast crisis (ly-BC; n=15) and pts with tyrosine kinase inhibitor (TKI)-resistant chronic phase harboring 2 or more BCR-ABL kinase domain mutations (mut-CP; n=9). Samples collected at diagnosis from CP pts (n=8) who achieved optimal response to therapy were also studied for comparison. A recent study in advanced systemic mastocytosis has demonstrated that SETD2 loss of function may result not only from genomic disruption but also from post-translational events, suggesting that the frequency of SETD2 involvement in solid tumors or hematologic malignancies might be higher than sequencing and copy number data alone would suggest. We thus decided to use a Western Blotting (WB) approach to assess H3K36Me3 levels as a surrogate marker of SETD2 loss of function and to screen for the expression of SETD2 full-length vs truncated protein isoforms, that are the most frequent result of inactivating nonsense or frameshift mutations. Reduced or null H3K36Me3 (as compared to a pool of healthy donors) was detected in the great majority of pts with advanced-phase CML (58/69, 84.1%; 12/15 AP, 24/30 my-BC, 12/15 ly-BC, 8/9 mut-CP). In contrast, CP pts at diagnosis showed H3K36Me3 levels superimposable to those of healthy donors. In some of them, however, the CD34+ cell fraction was available and reduced H3K36Me3 levels were detected in all cases. No evidence of abnormal SETD2 protein isoforms was detected, but pts with no H3K36Me3 also had no detectable SETD2 protein and pts with reduced H3K36Me3 had similarly reduced levels of full-length SETD2 protein as assessed by densitometric analysis of WB (Spearman R=0.95, p<0.001). SETD2 transcript levels, assessed by real time RT-PCR using GUSB as control gene, appeared not to be significantly lower in pts with no or low SETD2 expression as compared to healthy donors. Proteasomal inhibition in primary cells from pts with undetectable SETD2 by WB was found to rescue SETD2 expression and H3K36Me3 and to result in accumulation of hyper-ubiquitinated SETD2 protein.

To rule out the possibility that SETD2 loss of function might be another age-associated alteration in an epigenetic regulator, we collected a series of 80 healthy individuals with an age ranging from 65 to 95 years. In this cohort, we checked for SETD2 mutations by sequencing and for SETD2 and H3K36 trimethylation levels by WB. No mutations were detected and protein levels were not significantly lower in older adults than in a pool of younger adults.

Advanced-phase CML is characterized by a profound genetic heterogeneity. We here show the first evidence for a novel, nearly unifying alteration. However, SETD2 downmodulation and, consequently, H3K36Me3 deficiency seem to be most frequently accomplished at the post-translational level rather than being the results of genetic/genomic hits or transcriptional repression. Further studies aimed to assess whether H3K36Me3 loss might contribute to the genetic instability that is the hallmark of advanced-phase CML are warranted. Restoring physiological H3K36Me3 levels may help improve the outcome of this critical subset of pts.

Supported by AIL and AIRC (project code 16996)