Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas

Abstract

BackgroundHeterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown.MethodsClinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided.ResultshnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K’s oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients.ConclusionOur findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Alternative splicing and cancer: a systematic review

“… 129 However, another study found that hnRNPK is overexpressed in diffuse large B-cell lymphoma patients without Myc genomic alterations, representing a mechanism of C-Myc activation without Myc lesions, due to hnRNPK overexpression. 130 In addition, the overexpression of hnRNPK can promote lung metastasis, 131 and the increased hnRNPK expression is observed in multiple types of cancer, including breast, prostate, colorectal, gastric and pancreatic cancers. 132 Therefore, considering the inhibition of tumor cell growth by hnRNPK as a haploinsufficient tumor repressor in myeloid malignancies, 133 it is tempting to speculate that the dichotomous roles of hnRNPK in tumorigenesis may reflect the diverse hnRNPK functions in different cellular contexts.…”

Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing

“…Cells were homogenized in NP40 lysis buffer and standard immunoblotting procedures performed as previously described (Gallardo et al, 2019) using antibodies against hnRNP K (3C2), RUNX1 (EPR3099, both from Abcam, Cambridge, MA), β-actin (AC-15, Santa Cruz Biotechnology, Dallas, TX), Flag (F1804, Sigma Aldrich), HSP90 (ADI-SPA-836-D, Enzo Life Sciences)…”

“…As described previously (Gallardo et al, 2019), a computer-based algorithm was used that scan transcripts of interest for two or more (U/C)CCC motifs within 19 nucleotides.…”

“…Recombinant hnRNP K protein, produced in E.coli as described previously (Gallardo et al, 2019), was serially diluted in PBS (0.1 nM to 10 μM) and incubated with 6-FAM labelled RNA oligonucleotides. FA values were measured with excitation wavelength 485 nm and emission wavelength 528 nm on a Synergy Neo multi-mode plate reader (BioTek, Winooski, VT).…”

“…Overexpression of hnRNP K has been associated with adverse pathology in a handful of small clinical studies evaluating solid tumors (Barboro et al, 2009; Carpenter et al, 2006; Chen et al, 2010; Wen et al, 2010; Zhou et al, 2010). Further, elevated hnRNP K expression is not uncommon in B-cell lymphoma and has been defined as an oncogene in this setting (Gallardo et al, 2019). These findings led to consideration that hnRNP K aberrancies may contribute to a broader array of hematologic malignancies.…”