How many NOTCHes (activation pathways) are there in CLL? Free

In this issue of Blood, Guo et al¹ describe a novel mechanism of NOTCH1 pathway activation, involving the most frequent 3’ untranslated region (UTR) mutation (g.139390152, A>G), corresponding to a A>G mutation occurring 371 nucleotides downstream of the 7668 nucleotide-long NOTCH1 coding sequence.

The 3’UTR NOTCH1 mutations, overall found in 2% to 4% of patients with chronic lymphocytic leukemia (CLL), were firstly described by Puente et al,² who identified in data sets of whole-genome and whole-exome sequencing, several unusual splicing isoforms of NOTCH1, carrying somatic mutations in the 3’ UTR, including the position 7668+371A>G; these point mutations generate a novel motif recognized as a splicing acceptor, resulting in either exon 34 skipping or partial exon 34 deletion, both of which alter the NOTCH1 intracellular domain (NICD) terminal region.

Guo et al advanced the field by suggesting a novel paradigm that diverged from the classical model of NICD stabilization due to truncating mutations within exon 34; specifically, this 3’UTR splice variant, although turning out to be transcriptionally less active, is capable of dysregulating the physiological ubiquitination-dependent degradation of the wild-type NICD, resulting in an abnormal accumulation of unmutated NICD and the subsequent upregulation of the NOTCH1 pathway.

The story of NOTCH1 mutations in CLL continues to progress from the pivotal studies³ identifying NOTCH1 as one of the most frequently mutated genes at diagnosis, its association with poor outcome, and the increase of NOTCH1 mutational burden and frequency upon disease progression and Richter transformation.³ 

From a genomic point of view, all NOTCH1 mutations in CLL occur within exon 34, which encodes for about half of the NICD, specifically for the transactivation and the phosphodegron functional domains that are responsible for NOTCH1 transcriptional activity and stability. The most common mutation is a 2-base pair deletion occurring at position c.7541-7542 (NM_017617.5), which results in a premature stop codon. Functionally, these mutations ultimately end in the translation of a truncated protein, with the loss of the domains required for targeting of the NICD by ubiquitination and subsequent proteasomal degradation. Thus, the mutated protein fails to be ubiquitinated, displays an abnormal stability and is accumulated within the cell, prolonging the activation of NOTCH1 signaling.³ 

The study by Guo et al, although similarly involving the targeting of exon 34, suggests a completely different mechanism of NOTCH1 pathway activation. The authors provide evidence that the NOTCH1 variant, characterized by an altered C-terminus of 68 amino acids, rather than representing an abnormally stabilized NICD form, can act like a “molecular sponge” sequestering the proteins of the degradation machinery, thereby protecting the wild-type NICD and activating an abnormal NICD dependent signaling, thus justifying its oncogenic activity.

Curiously, the NOTCH1 activation mechanism proposed by Guo et al, closely resembles other mechanisms recently proposed in CLL, in which the abnormal accumulation of a normal NICD occurs as the result of mechanisms involving mutations in other genes. Loss-of-function mutations of genes involved in the phosphorylation of NICD, necessary for ubiquitin-based NICD degradation, like MED-12,⁴ or directly involved in ubiquitin binding, like FBXW7⁵ use this NOTCH1 activation mechanism. In both these situations, overall accounting for about 10% of CLL cases, an extended half-life of NICD is observed, thus mimicking the effects of canonical NOTCH1 mutations.

Similarly, loss-of-function mutations of the splicing factor SF3B1, found in about 10% of CLL, by altering the splicing of the DVL2 gene, that encodes for a mediator of the Wnt pathway and negative regulator of the NOTCH1 pathway, indirectly favors again the accumulation of a normal unmutated NICD and activation of the NOTCH1 signaling.⁶ 

Whatever the genetic mechanism(s) behind the NOTCH1 pathway activation, the functional consequences of the accumulation of an active NICD in CLL cells, can be summarized as causing enhanced CLL cell proliferation and survival mainly dependent on upregulation of MYC and MYC-related pathways, as well as triggering of the canonical nuclear factor-kB pathway.^7,8 Another phenotypic hallmark of NOTCH1 pathway activation in CLL is the lower expression of CD20 and the relative resistance of NOTCH1-mutated CLL cells to anti-CD20 exposure, due to a specific histone deacetylase–mediated epigenetic repression of the MS4A1/CD20 gene transcription by the accumulation of abnormally stable NICD in transcription complexes.⁹ 

In summary, multiple mechanisms, either NOTCH1 mutation dependent, including the novel one described by Guo et al, or NOTCH1 mutation independent, some of them already known, others still to be discovered, collectively contribute to identify the dysregulation of the NOTCH1 pathway as one of the major B-cell receptor–independent oncogenic events driving the expansion and shaping the phenotypic features of the CLL clone. In this regard, Fabbri et al¹⁰ estimated the aberrant accumulation of NICD in >50% of NOTCH1-unmutated CLL from peripheral blood. This aligns with earlier observations, quoted in Fabbri et al,¹⁰ of constitutively high NOTCH receptors (both NOTCH1 and NOTCH2) and ligand expression in CLL cells, suggesting that NOTCH signaling is a pervasive oncogenic driver beyond the mutational status. In this context, one can therefore speculate that the typically low expression of the CD20 antigen in CLL, which has been for long one of the main phenotypic CLL peculiarities recognized in CLL specific scores, could occur for mechanisms related to the activation of the NOTCH pathway.⁹ 

From a therapeutic standpoint, understanding the preferential strategies through which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL. As an example, Guo et al proposes targeting pathologically spliced mRNA with small molecules and antisense oligos to prevent the usage of alternative splice sites. Given the diversified landscape of mechanisms of NOTCH1 pathway activation summarized here, an alternative therapeutic strategy may be also the inhibition of downstream NOTCH signaling, independent of the pathogenic lesion, or the upstream blockage of the NOTCH receptor–ligand engagement.

In conclusion, Guo et al significantly enrich the diversity of genetic alterations converging on NOTCH1 pathway activation, revealing a novel splicing-dependent mechanism that complements known mutational landscapes and protein degradation defects. This work advances the conceptual framework of NOTCH1-driven oncogenesis and provides a foundation for novel therapeutic strategies in CLL.

Conflict-of-interest disclosure: The authors declare no competing financial interests.