Fig 2: Histological validation of early versus late cancer-associated fibroblast (CAF) signature. (A) Early- and late-stage PyMT tumors were harvested and analyzed for expression of the activated fibroblast marker, as well as the predicted early CAF marker Hexim1 and the late CAF marker Otx1 using PhenOptics. Nuclei were counterstained with DAPI. Representative images show combined expression of all markers as well as expression of single markers. Scale bars: 50 µm. (B) Hexim1 expression in total stromal cells is shown. (C) Otx1 expression in total stroma, αSMA+ stromal cells, and αSMA- stromal cells is displayed. Individual data points indicate mean expression of markers in tissue sections of one individual animal. Additionally, means ± SEM are shown. p-values were calculated using one-way ANOVA with Bonferroni’s correction, * p < 0.05, *** p < 0.001.

Fig 3: Model depicting the role of MEPCE in the 7SK snRNP complex and possible pathomechanism underlying the MEPCE nonsense variant. (a) In eukaryotes, synthesis of precursor mRNAs by RNA polymerase II (RNAP II) is essentially regulated by many factors (bottom figure). During the transcriptional process, RNAP II is paused proximal to the promoter by negative elongation factors (light and dark red ellipses). The release of RNAP II is mediated by phosphorylation through the positive transcription elongation factor b (P-TEFb) consisting of Cyclin-T1 (CT1) and the cyclin-dependent kinase 9 (CDK9). P-TEFb in turn is under control of the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex (top figure). The 7SK snRNP core complex consists of the 7SK snRNA that is permanently bound to MEPCE and LARP7. MEPCE stabilizes the 7SK snRNA by 5′ cap methylation, and LARP7 protects the 7SK snRNA by binding its 3′ end. P-TEFb inhibition and incorporation in the 7SK snRNP complex is ensued by HEXIM1/2 dimers. Upon extra- and/or intracellular stimuli, P-TEFb is released from this complex and recruited to the paused RNAP II. Here, the CDK9 subunit of P-TEFb phosphorylates negative elongation factors which then dissociate from the complex or are inactivated. These steps are necessary for the release of paused RNAP II. In addition, P-TEFb phosphorylates specific serines in the C-terminus of RNAP II to stimulate the elongation of transcription. (b) The MEPCE nonsense mutation leads to a decrease in MEPCE protein amount by ~50% in patient-derived cells that is accompanied by depletion of 7SK snRNA and LARP7 protein. Consequently, disintegration of the 7SK snRNP complex likely leads to enhanced release and activation of P-TEFb, followed by hyperphosphorylation of RNAP II’s C-terminal domain. We postulate that the P-TEFb equilibrium is shifted toward free P-TEFb in patient cells leading to preferred transition of RNAP II from the paused to the productively elongating state and dysregulated expression of P-TEFb-regulated genes.

Fig 4: Binding of HEXIM1 to the P-TEFb component Cyclin-T1 is reduced, and the C-terminal domain of RNAP II is hyperphosphorylated in patient cells. (a) Endogenous Cyclin-T1 was immunoprecipitated from lysates obtained from patient-, control 1- and patient’s mother-derived fibroblasts. For control purposes, lysates from all three samples were pooled and incubated with an IgG isotype control antibody (“Pool”). Immunoprecipitates (IP) and total cell lysates (Input) were analysed by immunoblotting using the indicated antibodies. The band representing the antibody heavy chain is indicated with a star. Representative blots are shown. Full-length blots are presented in Fig. S5. (b) Band intensities were quantified with a chemiluminescence imager. The amount of co-immunoprecipitated HEXIM1 was normalized to the precipitated amount of Cyclin-T1. The mean of five independent experiments ± SD is given. **p ≤ 0.01, ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (c) Immunoblot of lysates obtained from patient, control 1 and patient’s mother fibroblast cultures from three different passages. The amount of total and phosphorylated RNAP II (RNAP II-S2P) was monitored with specific antibodies, and anti-Tubulin antibody was used to control for equal loading. Representative blots are shown. Full-length blots are presented in Fig. S6. (d) Quantification of phosphorylated RNAP II. Band intensities were quantified with a chemiluminescence imager. The amount of RNAP II-S2P was normalized to total RNAP II. The mean of five independent experiments ± SD is given. ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison.

Fig 5: P-TEFb disruption from 7SK is dependent on the CTD but is independent of the BD domains. (A) Overexpression plasmids containing various full-length flag-tagged BRD4 constructs or (B) full-length and CTD-only constructs were transfected into 293T cells for 48 hours to determine the impact on HEXIM1 protein levels. (C) Overexpression constructs containing full-length WT, mutant, or the CTD domain alone were transfected into 293T cells for 48 hours and followed by a HEXIM1 or control IgG immunoprecipitation and western blot for associated proteins. Independent replicates for all blots are provided in Fig. S8.