The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness

Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

Dillon M. Voss1*, Anthony Sloan1*, Raffaella Spina, PhD2,3,4, Heather M. Ames, MD PhD2,4, and Eli E.

Bar, PhD2,3,4

1 Department of Neurological Surgery, Case Western Reserve University School of Medicine, Cleveland,

Ohio, United States of America.

2 Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United

States of America.

3 Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, United

States of America.

4 Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of

Medicine, Baltimore, Maryland, United States of America.

*Authors contributed equally to the study

Running Title: MBNL1 Regulates GBM Initiation and Progression

The authors declare no potential conflicts of interest.

Correspondence to:

Eli E. Bar, Ph.D.

Departments of Pathology and Neurosurgery, Marlene and Stewart Greenebaum Comprehensive Cancer

Center, University of Maryland School of Medicine 655 W. Baltimore St., Bressler Research Bldg.,

Room 8-039, Baltimore, MD 21201; [email protected] (office) 410-706-4826, (lab) 410-706-

2299

Word Count: 7946

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific regulators of RNA metabolism that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer glioblastoma (GBM) maintains stem-like features (GSC) through hypoxia- induced responses. Accordingly, we hypothesize here that hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSC rapidly exported MBNL1 out of the nucleus, resulting in significant inhibition of MBNL1 activity. Notably, hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression, resulting in significantly prolonged survival.

This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, where hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may therefore yield potent tumor suppressor activities, uncovering new therapeutic opportunities to counter this disease.

Significance: This study describes an unexpected mechanism by which RNA-binding protein

Muscleblind-Like-1(MBNL1) activity is inhibited in hypoxia by a simple isoform switch to regulate glioma stem cell self-renewal, tumorigenicity, and progression.

INTRODUCTION

Glioblastoma (GBM) is among the least curable cancers because of distinct sub-populations of proliferative and invasive cells that coordinately drive tumor growth, progression, and recurrence after therapy (1-3). The apparent genetic heterogeneity in GBM and other solid cancers, even within a single patient's cancer, demands the Identification and targeting of signaling nodes that are critical to multiple oncogenic pathways. Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in GBM (4). Over the past several years, we have demonstrated that hypoxia promotes the expansion of aggressive subpopulations of cells, often referred to as glioma stem cells (GSCs) (5). However, the mechanisms by which hypoxia regulates GSC are not entirely clear.

Oxygen levels play an essential role in regulating stem cells in multiple tissues, including the central nervous system (reviewed in (6)). Growth under low oxygen concentrations is known to maintain pluripotency and inhibit the differentiation of embryonic stem cells (7). In the rodent brain, hypoxia regulates self-renewal, survival, and differentiation of stem and progenitor cells (8, 9). In humans, hypoxia promotes the expansion of progenitor cells (10). We have recently shown that low oxygen levels commonly found in human tumors, promote the expansion of the cancer stem cell pool, as well as those grown in vitro for more extended periods (5). Along these lines, we and others have demonstrated the pivotal roles of hypoxia-inducible factors (HIFs) -1alpha and -2alpha in orchestrating the transcriptional and metabolic adaptations to hypoxia, and in inducing stem cell phenotypes in the hypoxic microenvironment ((5, 11-16), also reviewed in (17)). Thus, while the contributions of HIFs and hypoxia to promote self-renewal and expansion of glioma stem cells (GSCs) are established, the identity of additional regulators of GSC biology remains to be elucidated.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental process in the regulation of eukaryotic gene expression. The mammalian nervous system makes extensive use of splicing regulation to generate specialized protein isoforms that affect all aspects of neuronal development and function (18-

21). While splicing defects are increasingly implicated in neurological diseases and several types of cancer such as myelodysplastic syndrome and AML, a clear role for specific alternative splicing events in

GBM and their regulators remain to be established (22). Alternative splicing patterns are regulated by specialized RNA binding proteins that alter spliceosome assembly at specific splice sites (21, 23, 24). The

Muscle-Blind-Like (MBNL) family of RNA binding proteins have been studied extensively in the context of the neuromuscular disorder myotonic dystrophy, where inactivation of MBNL proteins result in a shift in splicing from an adult- to fetal-like patterns (25, 26).

Alternative mRNA splicing can substantially alter the functions of the proteins encoded by the mRNA (27). The Muscleblind-like (MBNL) family of sequence-specific pre-mRNA splicing factors bind

RNA through pairs of highly conserved zinc finger binding domains that recognize YGCY (where Y = C or U) and similar motifs (28-32). MBNL proteins are predominantly expressed in skeletal muscle, neuronal tissues, thymus, liver, and kidney and are essential for terminal differentiation of myocytes and neurons (33). In the brain, MBNL1 levels are significantly higher in astrocytes as compared with neural stem cells (34). MBNL1 is also involved in pluripotent stem cell differentiation, thereby linking isoform expression and the pluripotent and differentiation states (35). Importantly, Mbnl1 transcripts themselves undergo extensive alternative splicing, generating numerous protein isoforms. The inclusion of the highly conserved exon 5 is essential for nuclear localization and splicing activity of the MBNL1 protein (36,

37). The knockdown of Mbnl1 in cultured murine fetal liver progenitors blocks erythroid differentiation

(38). Inactivation of the MBNL1 protein is critical in the etiology of myotonic dystrophy, resulting in cataract formation, abnormal muscle relaxation, heart and nerve dysfunction, and other pathologies (25,

39). Importantly, MBNL1 inactivation results in an adult-to-fetal alternative splicing shift in numerous

MBNL1 target genes.

The function and expression of MBNL1 in gliomas and specifically in GSCs are currently unknown. So too is the role of the hypoxic GBM microenvironment in regulating MBNL1 activity.

Accordingly, this study explores the impact of hypoxia on MBNL1 activity in GSCs. In doing so, we

found that while MBNL1 is expressed in all gliomas, MBNL1 activity is inhibited in hypoxia, mimicking myotonic dystrophy-like adult-to-fetal alternative splicing switching in multiple MBNL1 target genes.

These included the fetal isoform of the GSC marker integrin alpha 6 (ITGA6) and CD47, a novel macrophage immune checkpoint protein that plays a broad role in cancer immune evasion across multiple cancer types, suggesting the involvement of MBNL1 in the regulation of GSC maintenance and immune evasion. From a therapeutic standpoint, we show that the active form of MBNL1 inhibits GSC self- renewal in vitro and tumorigenic potential in vivo and that inducing MBNL1 activity in established tumors can significantly prolong survival of animals bearing human GSC-derived orthotopic xenografts.

Our study not only reveals unexpected mechanisms by which MBNL1 activity is regulated by the hypoxic microenvironment but also demonstrates how hypoxia regulates cancer stem cell identity, GSC self- renewal, and tumorigenic potential by a simple isoform switch.

Materials and Methods

GBM Neurosphere Lines and Hypoxic Conditions

HSR-GBM1 and HSR-040821 and HSR-040622 were a kind gift from Dr. Angelo Vescovi and were established from freshly resected glioblastoma tumors and passaged as previously described (40). T387,

T3691, and T3832 were a kind gift from Dr. Jeremy N Rich. HSR-GBM1 and HSR040821 and HSR-

040622 are EGFR-WT, IDH1-WT. HSR-GBM1 is P53-WT, while HSR040821 carries an S278P point mutation in the P53 gene. The Phosphatase and Tensin homolog (PTEN) gene is WT in these three lines.

Transcriptional subtype classification: T387 is mesenchymal (41); T3691 and T3832 are proneural (42,

43). Similar averaged expression of classifier genes in HSR-GBM1, HSR040622, and HSR040821 neurosphere lines led to an inconclusive determination of subtype classification for these lines.

Mycoplasma testing was performed regularly, and the cultures were found to be negative. A hypoxic

° chamber maintained at 37 C, 1% O2, 5% CO2, and 94% N2 (X3 Biospherix) was used to conduct in vitro

hypoxic experiments. All hypoxic experiments were conducted on cells that were plated and allowed to recover overnight before hypoxic induction.

MBNL1 Depletion and Overexpression

GBM neurospheres were transduced with one of two lentiviruses expressing shRNA constructs

TRCN0000429155 and TRCN0000427657 (Sigma). We refer to these shRNA constructs as sh1 and sh2, respectively. Forty-eight hours later, cells were selected, in bulk, with 5g/ml puromycin (Thermo

Scientific) for three to seven days. In some experiments, the average of two independent cultures, each expressing one of the two shRNAs, is presented.

We refer to the active MBNL1 isoform that includes exon 5, only as MBNL1. The inactive isoform, lacking exon 5, is referred to as MBNL15. Lentivirus vector encoding for human MBNL1 under the constitutive CMV promoter was created by subcloning full-length MBNL1 cDNA from pCMV-

SPORT6/MBNL1 (TrueClone, ORIGENE) into pLenti6 using the Gateway cloning system (Invitrogen).

The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-

TRE>hMBNL1[NM_207293.1]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder

5 Inc.). All final constructs were sequenced either by the vendor or us. Generation of MBNL1 expressing lentivirus vector was achieved by site-directed mutagenesis, eliminating the coding sequence for exon five from the full-length cDNA in the pLV vector indicated above. Expression levels of MBNL1 and

MBNL15 mRNA and protein were determined by qPCR and Western blot analysis, respectively.

RNA isolation and qRT-PCR analysis

RNA was isolated from tissues and cells using the Quick-RNA isolation kit (Zymo Research). cDNA was generated by reverse transcription with a high-capacity cDNA reverse transcription kit with an RNase inhibitor (Thermo Fisher). For qPCR assays, gene expression analysis was performed using ten nanograms of cDNA and AmpliTaq Gold DNA Polymerase with SYBR Green (Thermo Fisher).

mRNA levels were normalized using Hypoxanthine Phosphoribosyltransferase 1 (HPRT). End-point RT–

PCR was performed using the Biorad C1000 Touch PCR Cycler and the following conditions. The first cycle of 8 min at 94 °C was followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. The reaction was ended with the extension step of 10 min at 72 °C. Visualization and analysis of amplified products were done using a Gel Doc XR System w/ Universal Hood II (Biorad) or an iBright FL1500 imaging system (Invitrogen). For the MBNL1 target genes, the amplified products were loaded on 2.0% agarose gel. Primer sequences are available upon request.

Immunofluorescence analysis

GBM neurospheres were cultured in normoxia or hypoxia for 48 hr. Neurospheres were fixed in 4% paraformaldehyde in PBS and incubated, at room temperature, for 5 min with occasional gentle mixing.

Following fixation, 200 l aliquots were "cytospun" against Premium Superfrost™ Microscope Slides

(Fisher Scientific 12-544-7) for 15 min at 1200 rpm. Flattened neurospheres were washed once with PBS containing 1M Glycine and two times with PBS. Following 2-min permeabilization step with 0.1% Triton

X-100 (in PBS) and incubation in PBS containing 0.1% bovine serum albumin. MBNL1 was revealed using primary monoclonal antibody MB1a(4A8) and secondary monkey-anti-mouse conjugated to Alexa fluor 488 (Invitrogen) and nuclei were stained using Hoechst 33342 (Sigma-Aldrich). Cells were washed in PBS and water, mounted with ProLong™ Diamond Antifade Mountant, and observed with a Nikon

W1 spinning disk confocal on Nikon Ti2 inverted microscope. Images were captured with a Hamamatsu sCMOS camera (Hamamatsu, Bridgewater, NJ).

Monoclonal antibody production

MB1a was deposited to the Developmental Studies Hybridoma Bank (DSHB) by Morris, G.E. (DSHB

Hybridoma Product MB1a(4A8)). Mouse MB1a hybridoma cells were purchased from DSHB and cultured in Iscove's Modified Dulbecco's Medium (IMDM, Sigma catalog # I3390) supplemented with

10% FBS, 0.1% Penicillin, 0.1% Gentamicin. For monoclonal antibody production, 1 x 106 cells,

suspended in 5 ml IMDM without serum, were inoculated into the cell compartment of a CELLine

(catalog # CL350) bioreactor. 340 ml of IMDM growth medium containing 10% FBS were included in the nutrient compartment. Supernatant was harvested once per week, where, in each cycle, one-fifth of the cells were inoculated back into the cell compartment, and the nutrient compartment was emptied, by aspiration, and filled with 340 ml of fresh growth medium. This cycle was repeated for seven times. The monoclonal MB1a (4A8) antibody was filtered through a 0.45 m syringe filter, and aliquotes were mixed with an equal volume of glycerol for long term storage at -20oC. Each batch of antibody was tested by Western blotting to confirm the presence of MBNL1 and MBNL15 isoforms in lysates of the 913 neurosphere line.

Immunoblot analysis

For whole-cell extracts, cells were lysed in buffer (50 mM Tris–HCl pH 8, 1% (w/v) NP-40, 1% (w/v)

SDS, 0.5% (w/v) Na-deoxycholate (DOC), 2 mM EDTA) supplemented with ‘protease inhibitor cocktail’

(Roche), incubated for 30 min at 4 °C, centrifuged for 30 min at 4 °C 16,000 g and the protein amounts were quantified using Pierce™ BCA Protein Assay Kit. Cell lysates were next sonicated and centrifuged at 4 °C 16,000 g. Supernatants were resolved by SDS–PAGE and transferred to nitrocellulose membranes using an iBlot™ 2 Gel Transfer Device (Invitrogen). Membranes were blocked in PBS containing 5%

(w/v) BSA and 0.05% (v/v) Tween-20 (PBST) for 1 hr and incubated with primary antibodies at 4 °C overnight. After washing with PBST, membranes were incubated with either anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 hr at room temperature. Signals were detected with an iBright FL1500 imaging system (Thermo Scientific), anti-MBNL1 (1:1000, see above), Anti-β-actin (1:20,000, AC-15 clone, Sigma), KPL Peroxidase-labeled antibody to mouse IgG

(1:7,500, 074-1806, SeraCare.com). MBNL1 immunohistochemical staining was performed using the monoclonal antibody at 1:200 concentration using a Ventana BenchMark Ultra. Immunostaining was optimized on de-identified normal human brain and glioblastoma specimens.

Bioinformatics and Gene Set Enrichment Analysis

Overall survival analysis of patients whose tumors are defined as mesenchymal (n=58) was performed on the Betastis portal: http://www.betastasis.com/glioma/tcga_gbm/. The portal censored three samples due to insufficient expression data.

We performed Gene set enrichment analysis (GSEA) according to (44). Normalized gene-level FPKM values for all samples were downloaded from the IvyGAP portal

(https://glioblastoma.alleninstitute.org/static/download.html). We compared all available 19 LE and 40

PAN samples available against the "cancer hallmarks" gene sets.

Most experiments were performed three or more times. Where appropriate, we included a bar graph representation of the average of independent experiments.

Finally, all animal studies were approved by CWRU IACUC. Protocol # 2018-0051

RESULTS

MBNL1 expression is highest in GBM defined as MES, inhibited in hypoxic elements of the tumor, and, within the MES subgroup, correlates with better overall patient survival.

Muscleblind-like-1 (MBNL1) is a crucial protein involved in the etiology of the RNA disease myotonic dystrophy. Its primary normal function is to modulate splicing during embryonic development (25, 32,

35, 38, 45-47). MBNL1 protein levels are low in pluripotent embryonic stem cells (ESC) and is dramatically increased in their differentiated progenies. This increase is accompanied by an MBNL1- regulated switch from a fetal-to-adult splicing pattern (15-17). A recent gene expression study of 1348

RNA-binding protein genes in 11 solid tumor types, together with splicing changes, has implicated

MBNL1 as a significant driver of splicing patterns in these tumors (48). To explore a potential role for

MBNL1 in GBM, we queried the TCGA database for MBNL1 expression in the four, transcriptionally

defined, GBM subtypes: classical (CL), proneural (PN), and mesenchymal (MES). This analysis showed that MBNL1 is expressed in all GBMs. In CL and PN GBMs, MBNL1 levels were 17-22% over healthy brain controls. However, this finding was found not to reach statistical significance.

In contrast, we found that MES tumors express, on average, 50% higher levels of MBNL1 mRNA, and this was found to be highly significant (Figure 1A). Next, we queried the Ivy Glioblastoma Atlas Project

(IVYGAP) RNAseq database to identify differences in MBNL1 expression in different anatomical structures of GBM defined by reference histology. To this end, we focused on the leading edge (LE), which is generally accepted to contain primarily normoxic cells, and on pseudopallisading cells around centers of necrosis (PAN), which we have previously suggested to contain the hypoxic cancer stem cell niche (17). Gene set enrichment analysis (GSEA, (44)) comparing PAN to LE further confirmed that the hypoxia gene-set is significantly enriched in PAN as compared with LE (Figure 1B). A heatmap of the entire hypoxia gene-set (Figure 1B, right) shows that the vast majority of the genes in this gene-set are overexpressed in PAN. Also, and consistent with our recent publication showing that mTOR signaling is induced in hypoxic GBM neurospheres (16), we found the mTOR gene-set to be similarly enriched in

PAN (Figure 1B). MBNL1 mRNA levels were significantly downregulated in PAN as compared to the

LE (Figure 1C). To determine if reduced MBNL1 expression in hypoxia is a property of a specific subtype or shared among the subtypes, we further queried MBNL1 expression in LE and PAN by subtype. We found that significant decreases in MBNL1 mRNA levels were primarily the property of mesenchymal (Figure 1D) and classical (Supplementary Figure S1) tumors. Taken together, these data show that MBNL1 is expressed in all GBMs and that in the majority of GBMs, MBNL1 mRNA expression is lower in hypoxic elements of the tumor. To determine the potential clinical implications of reduced MBNL1 expression in this area, we queried the TCGA database focusing on patient survival by

GBM subtype. The analysis revealed that reduced MBNL1 expression is associated with worse patient survival (8.7 vs. 14.3 months), and this was found to be the exclusive property of tumors, which are defined as mesenchymal subtype (Figure 1E).

Hypoxia inhibits MBNL1 splicing activity.

Next, we determined MBNL1 mRNA expression levels in snap-frozen tissues from numerous GBM neurosphere-derived xenograft models by quantitative real-time PCR (qRT-PCR). We found MBNL1 levels were variable ranging from 0.39 to 1.8 the level of the house-keeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Figure 2A). It has been recently shown that GBM subtype, determined based on "bulk" RNA analyses, is the property of individual cells. Each tumor contains cells, transcriptionally defined as MES, PN, or CL (49). Furthermore, in a more recent study, Neftel and colleagues have shown that transcriptional subtype classification can be defined as a "state" rather than as a fixed identity and that cells may transition between "states" (50). To determine if MBNL1 expression is associated with a particular state (namely, OPC-like, NPC-like, AC-like, or MES-like), we queried the database described by Neftel and colleagues (https://singlecell.broadinstitute.org/single_cell/study/

SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma) and found that MBNL1 is expressed in all four "states" (Figure 2B). In cultured neurospheres, MBNL1 protein levels were variable as well and were not significantly affected by hypoxia (Figure 2C). It is well documented that the splicing activity of

MBNL1 is regulated in a negative feedback loop. MBNL1 promotes "self-splicing" of the MBNL1 pre- mRNA to generate a shorter isoform that lacks the highly conserved exon 5 (MBNL15), encoding for 18 amino acids that are C-terminal of the fourth and final zinc finger RNA binding domain (Figure 2D).

Inhibition of MBNL1 results in the generation of an MBNL1 isoform that includes exon 5 (MBNL1).

Therefore a delicate balance between MBNL1 and MBNL15 isoforms control the overall MBNL1 activity (36). We, therefore, used Western blot analysis to quantify relative MBNL1 protein isoform expression levels. In healthy human brain tissues (NB1, NB2), MBNL15 is by far the dominant isoform representing 94% and 93% of total MBNL1 protein (Figure 2E). In 913 xenografts implanted in the flank

(fl) or orthotopically (br), we documented a significant amount of the MBNL1 isoform, representing 38% and 25% of total MBNL1 protein, respectively. RT-PCR analysis of MBNL1 mRNA isoforms in GBM neurosphere-initiated xenografts consistently showed both MBNL1 and MBNL15 isoform transcripts are

expressed in vivo. In contrast, and consistent with Figure 2E, we noted that in healthy brain tissues,

MBNL15 mRNA is expressed almost exclusively (Figure 2F). To determine if MBNL1 expression in vivo may be due to hypoxia, we cultured 913 and 821 neurospheres in normoxia or hypoxia followed by

Western blot to analyze MBNL1 protein isoform expression. Indeed, we found that hypoxia promoted a relative increase of MBNL1 over MBNL15 from 56% to 74% and from 37% to 61%, in 913 and 821 neurospheres, respectively (Figure 2G). Since MBNL1 protein promotes the splicing of its pre-mRNA

(36), this data suggests that hypoxia regulates MBNL1 splicing activity. To test this directly, we examined mRNA splicing patterns of five genes previously shown to be directly regulated by MBNL1 in human embryonic stem cells (hESC)(35). Importantly, the functional consequence of MBNL1 binding to pre-mRNAs is known to result in either inclusion or exclusion of the downstream exon. For example, while MBNL1 binding to the MBNL1 and CD47 pre-mRNAs results in the exclusion of the downstream exon, MBNL1 binding to ITGA6, CTTN, and CLSTN1 pre-mRNAs promotes the inclusion of the downstream exon. We found dramatic switch-like shifts in the splicing patterns of all five genes from the differentiated (D) to undifferentiated (S), embryonic stem cell-like, states. Representative data from 913 neurospheres is shown in Figure 2H and the average of three independent experiments is shown in Figure

2I. These changes are consistent with loss of MBNL1 activity as they mirror the results obtained when

MBNL1 expression was knocked down with short hairpin RNAs (Figure 2I, purple bars, and

Supplementary Figure S2 (A-B). Similar results were obtained with 622 and 821 neurosphere lines and are shown in Supplementary Figure S3. In contrast, an analysis of 08-387, 3691, and 3832 neurosphere lines showed no significant changes in MBNL1 activity. Expression of the MBNL15 isoform did not affect the splicing of MBNL1 pre-mRNA targets (Figure 2J), confirming that pre-mRNA splicing is facilitated by the MBNL1 isoform. Together, these results show that while MBNL1 mRNA and protein levels are variable in GBMs, hypoxia inhibits MBNL1 activity in some tumor-derived neurosphere lines to promote the expression of numerous gene isoforms associated with an embryonic stem cell-like state.

A bichromatic fluorescent reporter for MBNL-directed alternative splicing confirms hypoxia- dependent inhibition of MBNL1 splicing activity at the single-cell level.

To measure MBNL1 activity at the single-cell level, we utilized a bichromatic fluorescent reporter for

MBNL-directed alternative splicing. This reporter contains an artificial exon of 28 nt, responsive to

MBNL1, that shifts the reading frame from dsRED (exon skipping) to GFP (exon inclusion) as described previously (51) and schematically illustrated in Figure 3A. In this case, the expression of dsRed is indicative of MBNL1 activity, while GFP represents inactivity. To validate the reporter faithfully reports

MBNL1 activity in GSC, we first examined the 913 neurosphere line, electroporated with the RG6 reporter, by imaging flow cytometry (Figure 3B) and documented the presence of sorted dsREDHIGH and dsREDLOW subpopulations. To test if the dsREDHIGH subpopulation is enriched in active MBNL1 protein, we electroporated the 913 neurosphere line with the RG6 reporter and FACS sorted dsREDHIGH and dsREDLOW cells and analyzed the splicing pattern of MBNL1 target genes (Supplementary Figure S4A,

B). This set of experiments confirmed that MBNL1 activity is significantly higher in dsREDHIGH as compared with dsREDLOW cells. To determine if MBNL1 is required for splicing bias towards dsRED, we electroporated the 913 neurosphere line, integrating control or one of two shMBNL1 expressing lentiviruses (sh1 or sh2) with the RG6 reporter. We found that reduced MBNL1 expression resulted in a significant reduction in the dsREDHIGH population, indicating that MBNL1 is indeed required for splicing of the reporter to generate dsRED (Supplementary Figure S4C). To measure the effects of hypoxia on

MBNL1 activity, we cultured the 913 neurosphere line, electroporated with the RG6 reporter, in normoxia or hypoxia for 24 hours and determined MBNL1 activity by flow cytometry. This analysis showed that when cultured in normoxia, the 913 neurospheres had an almost even distribution of dsREDHIGH and dsREDLOW populations (Figure 3C). In contrast, cells cultured in hypoxia showed an

87.5% inhibition of the dsREDHIGH population with a substantial increase from 52% to 94% in the dsREDLOW sub-population (Figure 3D). This set of experiments provides further support to the "bulk" analyses described in Figure 2 and indicates that each GBM cell expresses both MBNL1 isoforms in normoxia, but under hypoxic conditions, MBNL1 activity is rapidly inhibited in most cells.

Hypoxia promotes MBNL1 nuclear export.

Given the rapid inactivation of MBNL1 splicing activity we observed in hypoxic GBM neurospheres, we next determined if this process may involve nuclear export of MBNL1. To this end, we cultured GBM neurospheres in normoxia and hypoxia, followed by immunostaining for MBNL1. Remarkably, we found that MBNL1 localizes to the cytoplasm and nucleus of GBM neurospheres, which are cultured in normoxia. In contrast, MBNL1 is rapidly exported out of the nucleus under hypoxic conditions. These results clearly show that the hypoxia-dependent inactivation of MBNL1 splicing activity involves

MBNL1 nuclear export (Figure 3E-F). Finally, to determine if MBNL1 nuclear exclusion occurs in vivo, we immunohistochemically stained human GBM surgical sections for MBNL1. As shown in Figure 3G, cells with robust nuclear localization and with predominantly cytoplasmic localization are found in these specimens confirming that nuclear-cytoplasmic transport occurs in vivo.

MBNL1 inhibits GSC self-renewal in vitro.

Because MBNL1 activity is required for differentiation of ESCs (35), we next asked whether MBNL1 may inhibit GSC self-renewal, one of the fundamental properties of all stem cells. Because self-renewal and proliferation are linked, we first determined the effect of MBNL1 expression of neurosphere proliferation. Supplementary Figure S5 shows that conditional expression of MBNL1-mCherry

(Supplementary S5A) results in a small but significant reduction of cell viability (Supplementary Figure

S5B) and that this translates to a significant decrease in proliferation (Supplementary Figure S5C). To determine the effect of MBNL1 on the self-renewal capacity of GBM neurospheres in a quantitative fashion, we transiently electroporated plasmids encoding for human MBNL1 or GFP, as control, under the control of a constitutive promoter. Cells were recovered overnight before being challenged in a self- renewal assay in methylcellulose for ten days, as we previously described (5). We found that transient expression of MBNL1 significantly reduced GSC self-renewal in most neurosphere lines. Specifically,

MBNL1 reduced the average sphere diameter of the 913 neurosphere line from 162.9 m to 109.9 m

(student t-test *** p<0.0001; Figure 4A). Similar reductions were documented in 821 (164.1 m to 106.0

m; student t-test *** p<0.0001; Figure 4B), 3691 (159.8 m to 102.3 m; student t-test *** p<0.0001;

Figure 4C), and 3832 neurosphere lines (154.8 m to 95.5 m; student t-test *** p<0.0001; Figure 4D). A similar trend, albeit less robust, was documented in 622 and 08-387 neurosphere lines (Supplementary

Figure S6).

MBNL1 inhibits GBM tumor initiation and progression

Next, we tested the effect of MBNL1 on tumor initiation and progression using a constitutive and doxycycline-inducible system approaches, respectively. Quantitative real-time PCR analysis confirmed a modest fourfold increase in MBNL1 mRNA expression levels in 913 neurospheres transduced with pLenti6-MBNL1 compared with vector control (Figure 5A). Tumor initiation, as detected by bioluminescence imaging, was confirmed in the control group as early as 23 days post-injection. In contrast, it took cells transduced with lentivirus expressing MBNL1 roughly 80 days for luciferase activity to cross the radiance threshold of 1x106 (Figure 5B). Representative animals from this experiment are shown in Figure 5C. This delay in tumor initiation affected median survival (MS). Animals bearing xenografts transduced with pLenti6 lentivirus had a median survival of 58 days compared with over 200 days for MBNL1 expressing tumors (Figure 5D; log-rank survival analysis ** P<0.01).

Next, we sought to determine the effects of MBNL1 on the progression of established tumors. To this end, we transduced the 913 neurosphere line with lentiviruses expressing MBNL1-mCherry under the control of a tetracycline-inducible system (913 MBNL1-mCherry Tet ON). We found that doxycycline- dependent induction of MBNL1-mCherry occurs in a dose-dependent fashion (Figure 5E). Flow cytometry was used to determine the percentage and magnitude of this induction at the single-cell level.

The percentage of mCherry-positive cells increased from 1.1% to roughly 60% in the vehicle and doxycycline-treated 913 MBNL1-mCherry Tet ON neurospheres, respectively. Also, we found roughly a

six-fold increase in the fluorescent intensity, over the background, in neurospheres treated with doxycycline (Figure 5F, G). Next, we determined the activity of doxycycline-induced MBNL1-mCherry by examining the splicing patterns of MBNL1 target genes (Figure 5H) and its effects on GSC self- renewal (Supplementary Figure S7A). Supplementary Figure S7B shows self-renewal analysis of control cells, expressing rtTA but not MBNL1 (913 Luciferase Control Tet ON).

913 Luciferase MBNL1-mCherry Tet ON and 913 Luciferase rtTA-control cultures were also used to determine the effects of MBNL1 on tumor progression. Following the establishment of large orthotopic xenografts, where Radiance exceeded 1 x 106, mice were randomized to control and doxycycline treatment groups. Each group included four females and four males. We found that doxycycline induced MBNL1 expression in tumors significantly prolonged the survival of animals

(Figure 5I). At the end of the experiment, 0 of 8 and 7 of 8 animals were alive in the control and doxycycline treatment groups, respectively. In contrast, and consistent with the in vitro self-renewal assays (Supplementary Figure S7), doxycycline-treated animals bearing tumors integrating only rtTA had similar median survival (59.5 versus 63.5 days) to their control, non-induced, counterparts

(Supplementary Figure S8).

DISCUSSION

The vast majority of human genes are alternatively spliced, generating RNA isoforms that code for functionally distinct proteins (52, 53). Several recent reports highlight genes that are alternatively spliced in response to hypoxia. However, very few studies address the molecular mechanism regulating splicing in hypoxia (reviewed in (54)). Initial in silico analyses provided support to the notion that while the splicing regulator MBNL1 is expressed in all GBM and the vast majority of the cells in each tumor, as evidenced by single-cell RNAseq, generally reduced expression of MBNL1 correlated with worse overall survival of patients. Interestingly, this association was found to be the exclusive property of tumors

transcriptionally defined as mesenchymal. Taken together with the knowledge that MBNL1 promotes cellular differentiation during embryonic development (35), we hypothesized that MBNL1 might act as a tumor suppressor and thus explored the possibility that its activity may also be regulated. Indeed, we show that healthy brain tissues primarily express the short MBNL15 isoform, representing the

"differentiated" state.

In contrast, GBMs express a mix of the "embryonic" and the "differentiated" state isoforms.

Importantly, when GBM neurospheres are cultured in hypoxia, they mostly express the "embryonic" mRNA isoform of MBNL1, which is indicative of MBNL1 protein inactivation (Figure 2). This inactivation is readily detectable at the single-cell level (Figure 3). Here, the bichromatic RG6 MBNL1 splicing minigene proved to be a sensitive measure of MBNL1-dependent splicing. Also, confocal microscopy performed on GBM neurospheres cultured in normoxia and hypoxia provided evidence for hypoxia dependent MBNL1 inactivation by MBNL1 nuclear export. It would be necessary, in future studies, to elucidate this transport mechanism. These results show that while MBNL1 mRNA and protein are expressed in all GBMs and most cells within each tumor, the activity of MBNL1 is rapidly inhibited in hypoxia and that in tumors, reduced MBNL1 expression is associated with worse patient outcome.

Finally, we showed, for the first time, that the MBNL1 protein is localized to the cytoplasm or nucleus in human GBM surgical specimens (Figure 3G). These data suggest that cells may require to expel MBNL1 out of the nucleus under certain physiological conditions. We show that hypoxia promotes the export of

MBNL1 out of the nucleus, but we would like to speculate that other stress conditions such as acidic stress or inflammation may also require cells to export MBNL1 out of the nucleus. These are all areas of interest that we continue to investigate.

If MBNL1 activity is inhibited in GBM to promote a stem cell state, would forced expression of the active isoform of MBNL1 inhibit tumor growth? Also, if MBNL1 expression is forced upon GSCs, will they exhibit reduced clonogenic and tumorigenic capacities? Indeed, we found that lentivirus expression of the pro-differentiation, active, isoform of MBNL1 significantly inhibited GSC self-renewal

of multiple GBM neurosphere lines (Figure 4). Importantly, these effects were documented even in GBM neurosphere lines were refractory to hypoxia in the context of MBNL1 splicing activity, indicating that most GBM may share a sensitivity to MBNL1 activity. Furthermore, MBNL1 blocked tumor initiation and progression in orthotopic GSC-derived xenografts indicating that the pro-differentiation isoform of

MBNL1 is tumor suppressive (Figure 5).

It has been recently reported that in breast cancer, MBNL1 suppresses metastatic colonization and stabilizes metastasis suppressor transcripts (55). While we have not observed apparent effects of MBNL1 expression on cell invasion in vivo, this aspect of MBNL1 activity remains to be explored in future studies. Also, a recent study by Fischer and colleagues described the role of MBNL2 in controlling the transcript abundance of hypoxia response genes in lung and breast carcinomas (56). We could not detect significant changes in MBNL2 mRNA levels in hypoxia in the models we studied. Our study reveals, for the first time, a novel and essential role for MBNL1 in GSC maintenance and links the hypoxic microenvironment to the regulation of alternative RNA splicing. Future studies will be required to identify the signaling pathways which are regulated by MBNL1 directed alternative RNA splicing.

We propose that a signaling pathway, activated in hypoxic cells, inhibits MBNL1 activity, by nuclear export, to induce the stem cell state. While the identity of this inhibitory pathway remains to be uncovered, therapies which are aimed at reversing the inhibition of MBNL1 splicing activity in GBM, and likely other tumors, may result in a new therapeutic avenue for these untreatable cancers.

Acknowledgements

Funding: NIH NINDS R21NS106553, NCI R01CA187780

Authorship: E.E.B, D.V.M, A.S, R.S, H.M.A, designed, and or performed experiments. E.E.B, R.S,

H.M.A, interpreted data. E.E.B drafted the manuscript.

References

1. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell. 2006;9(5):391-403. 2. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63(18):5821-8. 3. Lathia JD, Mack SC, Mulkearns-Hubert EE, Valentim CL, and Rich JN. Cancer stem cells in glioblastoma. Genes Dev. 2015;29(12):1203-17. 4. Komori T, Sasaki H, and Yoshida K. [Revised WHO Classification of Tumours of the Central Nervous System:Summary of the Revision and Perspective]. No Shinkei Geka. 2016;44(8):625- 35. 5. Bar EE, Lin A, Mahairaki V, Matsui W, and Eberhart CG. Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres. Am J Pathol. 2010;177(3):1491-502. 6. Panchision DM. The role of oxygen in regulating neural stem cells in development and disease. J Cell Physiol. 2009;220(3):562-8. 7. Ezashi T, Das P, and Roberts RM. Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A. 2005;102(13):4783-8. 8. Studer L, Csete M, Lee SH, Kabbani N, Walikonis J, Wold B, et al. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci. 2000;20(19):7377-83. 9. Morrison SJ, Csete M, Groves AK, Melega W, Wold B, and Anderson DJ. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J Neurosci. 2000;20(19):7370-6. 10. Pistollato F, Chen HL, Schwartz PH, Basso G, and Panchision DM. Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes. Mol Cell Neurosci. 2007;35(3):424-35. 11. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, and Rich JN. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle. 2009;8(20):3274-84.

12. Pistollato F, Chen HL, Rood BR, Zhang HZ, D'Avella D, Denaro L, et al. Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma. Stem Cells. 2009;27(1):7-17. 13. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell. 2009;15(6):501-13. 14. Seidel S, Garvalov BK, Wirta V, von Stechow L, Schanzer A, Meletis K, et al. A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain. 2010;133(Pt 4):983-95. 15. Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis A, McKay RD, et al. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene. 2009;28(45):3949-59. 16. Spina R, Voss DM, Yang X, Sohn JW, Vinkler R, Schraner J, et al. MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner. Neurooncol Adv. 2020;2(1):vdz062. 17. Bar EE. Glioblastoma, cancer stem cells and hypoxia. Brain Pathol. 2011;21(2):119-29. 18. Li Q, Lee JA, and Black DL. Neuronal regulation of alternative pre-mRNA splicing. Nat Rev Neurosci. 2007;8(11):819-31. 19. Zheng S, and Black DL. Alternative pre-mRNA splicing in neurons: growing up and extending its reach. Trends Genet. 2013;29(8):442-8. 20. Raj B, and Blencowe BJ. Alternative Splicing in the Mammalian Nervous System: Recent Insights into Mechanisms and Functional Roles. Neuron. 2015;87(1):14-27. 21. Will CL, and Luhrmann R. Spliceosome structure and function. Cold Spring Harb Perspect Biol. 2011;3(7). 22. Visconte V, M ON, and H JR. Mutations in Splicing Factor Genes in Myeloid Malignancies: Significance and Impact on Clinical Features. Cancers (Basel). 2019;11(12). 23. Fu XD, and Ares M, Jr. Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet. 2014;15(10):689-701. 24. Lee Y, and Rio DC. Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annu Rev Biochem. 2015;84:291-323. 25. Lin X, Miller JW, Mankodi A, Kanadia RN, Yuan Y, Moxley RT, et al. Failure of MBNL1- dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet. 2006;15(13):2087-97. 26. Kuyumcu-Martinez NM, and Cooper TA. Misregulation of alternative splicing causes pathogenesis in myotonic dystrophy. Prog Mol Subcell Biol. 2006;44:133-59. 27. Maniatis T, and Tasic B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature. 2002;418(6894):236-43. 28. Begemann G, Paricio N, Artero R, Kiss I, Perez-Alonso M, and Mlodzik M. muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. Development. 1997;124(21):4321-31. 29. Teplova M, and Patel DJ. Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1. Nat Struct Mol Biol. 2008;15(12):1343-51. 30. Ho TH, Charlet BN, Poulos MG, Singh G, Swanson MS, and Cooper TA. Muscleblind proteins regulate alternative splicing. EMBO J. 2004;23(15):3103-12. 31. Fernandez-Costa JM, Llamusi MB, Garcia-Lopez A, and Artero R. Alternative splicing regulation by Muscleblind proteins: from development to disease. Biol Rev Camb Philos Soc. 2011;86(4):947-58. 32. Wang ET, Cody NA, Jog S, Biancolella M, Wang TT, Treacy DJ, et al. Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins. Cell. 2012;150(4):710-24.

33. Kanadia RN, Urbinati CR, Crusselle VJ, Luo D, Lee YJ, Harrison JK, et al. Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3. Gene Expr Patterns. 2003;3(4):459-62. 34. Malik N, Wang X, Shah S, Efthymiou AG, Yan B, Heman-Ackah S, et al. Comparison of the gene expression profiles of human fetal cortical astrocytes with pluripotent stem cell derived neural stem cells identifies human astrocyte markers and signaling pathways and transcription factors active in human astrocytes. PLoS One. 2014;9(5):e96139. 35. Venables JP, Lapasset L, Gadea G, Fort P, Klinck R, Irimia M, et al. MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation. Nat Commun. 2013;4:2480. 36. Gates DP, Coonrod LA, and Berglund JA. Autoregulated splicing of muscleblind-like 1 (MBNL1) Pre-mRNA. J Biol Chem. 2011;286(39):34224-33. 37. Miller JW, Urbinati CR, Teng-Umnuay P, Stenberg MG, Byrne BJ, Thornton CA, et al. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J. 2000;19(17):4439-48. 38. Cheng AW, Shi J, Wong P, Luo KL, Trepman P, Wang ET, et al. Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis. Blood. 2014;124(4):598- 610. 39. Kanadia RN, Johnstone KA, Mankodi A, Lungu C, Thornton CA, Esson D, et al. A muscleblind knockout model for myotonic dystrophy. Science. 2003;302(5652):1978-80. 40. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004;64(19):7011-21. 41. Alvarado AG, Thiagarajan PS, Mulkearns-Hubert EE, Silver DJ, Hale JS, Alban TJ, et al. Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression. Cell Stem Cell. 2017;20(4):450-61 e4. 42. Madhankumar AB, Mrowczynski O, Slagle-Webb B, Thomas A, Zacharia B, Mintz A, et al. TMIC-02. INTERACTION OF LIGAND CONJUGATED QUANTUM DOTS WITH THE GLIOMA STEM CELL SECRETED EXOSOMES AND SUBSEQUENT UPTAKE BY THE GLIOMA STEM CELLS OF VARIOUS SUBTYPES. Neuro-Oncology. 2018;20(suppl_6):vi256-vi. 43. Jung J, Kim LJ, Wang X, Wu Q, Sanvoranart T, Hubert CG, et al. Nicotinamide metabolism regulates glioblastoma stem cell maintenance. JCI Insight. 2017;2(10). 44. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-50. 45. Rinaldi F, Terracciano C, Pisani V, Massa R, Loro E, Vergani L, et al. Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues. Neurobiol Dis. 2012;45(1):264-71. 46. Kalsotra A, Xiao X, Ward AJ, Castle JC, Johnson JM, Burge CB, et al. Proc Natl Acad Sci U S A. United States; 2008:20333-8. 47. Fugier C, Klein AF, Hammer C, Vassilopoulos S, Ivarsson Y, Toussaint A, et al. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. Nat Med. 2011;17(6):720-5. 48. Sebestyen E, Singh B, Minana B, Pages A, Mateo F, Pujana MA, et al. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. Genome Res. 2016;26(6):732-44. 49. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Wakimoto H, et al. Single-cell RNA- seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014;344(6190):1396-401.

50. Neftel C, Laffy J, Filbin MG, Hara T, Shore ME, Rahme GJ, et al. An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma. Cell. 2019;178(4):835-49 e21. 51. Orengo JP, Bundman D, and Cooper TA. A bichromatic fluorescent reporter for cell-based screens of alternative splicing. Nucleic Acids Res. 2006;34(22):e148. 52. Cooper TA, Wan L, and Dreyfuss G. RNA and disease. Cell. 2009;136(4):777-93. 53. Philips AV, and Cooper TA. RNA processing and human disease. Cell Mol Life Sci. 2000;57(2):235-49. 54. Kanopka A. Cell survival: Interplay between hypoxia and pre-mRNA splicing. Exp Cell Res. 2017;356(2):187-91. 55. Fish L, Pencheva N, Goodarzi H, Tran H, Yoshida M, and Tavazoie SF. Muscleblind-like 1 suppresses breast cancer metastatic colonization and stabilizes metastasis suppressor transcripts. Genes Dev. 2016;30(4):386-98. 56. Fischer S, Di Liddo A, Taylor K, Gerhardus JS, Sobczak K, Zarnack K, et al. Muscleblind-like 2 controls the hypoxia response of cancer cells. RNA. 2020;26(5):648-63.

Figure Legends

Figure 1. MBNL1 expression in glioblastoma subtypes and its relation to patient survival. (A) MBNL1 mRNA levels in normal cortex and the three, transcriptionally defined, glioblastoma subtypes (CL - classical, PN - Proneural, MES - Mesenchymal). Expression levels are derived from publicly available Affymetrix HT HG U133A data (The Cancer Genome Atlas (http://cancergenome.nih.gov). Statistics: one-way ANOVA **** p<0.0001. (B) Gene-set-enrichment (GSEA) analysis. Pseudopallisading cells around centers of necrosis (PAN) are enriched for the hypoxia (top) and MTORC1 (bottom) gene sets. Normalized Enrichment Score (NES). Nominal p-value (P). The heatmap, on the right, shows the expression level of hypoxia signature genes in PAN and LE. (C) MBNL1 expression in PAN and LE in all GBMs (student t-test, * p<0.05). (D) MBNL1 expression in mesenchymal GBM (student t-test ** p<0.01). (E) Kaplan–Meier survival analysis, using TCGA data, shows that low expression of MBNL1 in mesenchymal GBM can predict shorter overall survival (8.7 vs. 14.3 months, log-rank test **** p=0.00234). Project Betastasis.

Figure 2. MBNL1 expression in GBM xenografts and cultured GSCs. (A) Quantitative real-time PCR analysis of MBNL1 mRNA expression in GSC-derived xenografts. (B) Two-dimensional representation of MBNL1 expression in GBM cellular states. (C) MBNL1 protein (total) expression in 913 and 821 neurospheres cultured in normoxia (21% oxygen) or hypoxia (1% oxygen). Actin was used for normalization. (D) A cartoon illustrating the MBNL1 gene with exon 5 being the target of MBNL1 directed alternative splicing resulting in the generation of the MBNL1Δ5 isoform. Inactivation of MBNL1 in hypoxia, defaults to the MBNL1 isoform (E) Western blot for MBNL1 isoform expression in non-neoplastic brains (NB1, NB2) and 913 xenografts implanted in the flank (fl) and brain (br). Exon 5 inclusion is indicated below. (F) MBNL1 isoform expression in the normal brain and six GBM neurosphere lines. (G) Western blot for MBNL1 isoform expression in 913 and 821 neurospheres cultured in normoxia or hypoxia. Exon 5 inclusion is indicated below. (H) MBNL1 target genes ITGA6, CD47, CTTN, CLSTN1, and MBNL1 splicing pattern in 913 neurospheres cultured in normoxia and hypoxia. S and D bands correspond to products amplified from ESCs and their differentiated progenies, respectively (described in Venables et al., 2013). (I) Bar graphs summarizing the results of three independent experiments calculating percent exon exclusion in each of the five MBNL1 target genes in normoxia, hypoxia, and GBM neurospheres expressing shMBNL1 (average of two independent shRNAs). Similar analyses were performed on 622, and 821 GSC lines showed similar results, the results from 913 neurospheres are shown. (J) Splicing of MBNL1 target genes is unaffected

by MBNL1Δ5 as compared with parental neurospheres (C) and neurospheres transduced with empty vector (V) as controls. Statistical analyses: one-way ANOVA. ** P<0.01, * P<0.05. In Western blots, actin was used as a loading control and for calculating relative isoform expression.

Figure 3. Bichromatic alternative splicing reporter confirms MBNL1 activity is inhibited in hypoxia. (A) Diagram of the RG6 minigene. AS of a 28nt cassette exon, responsive to MBNL1, shifts the reading frame between dsRED and GFP (adapted from Orengo et al., 2006). (B) Imaging flow cytometry confirms the presence of three populations expressing high, intermediate, and low MBNL1 activity in GSCs. (C-D) MBNL1 activity is inhibited in hypoxia, as indicated by an almost complete shift towards GFP expression (exon inclusion). (E-F) Hypoxia (1% oxygen) promotes MBNL1 nuclear export - providing spatial support for the inactivation of MBNL1 in hypoxia. (G) Nuclear and cytoplasmic localization of MBNL1 in human glioblastoma. (i) Representative human glioblastoma surgical section (scale bar = 10m) immunohistochemically stained for MBNL1. Cells with robust nuclear localization (solid outline) and with predominantly cytoplasmic localization (dashed outline) are magnified an additional 2.5X in the (ii) and (iii) panels, respectively.

Figure 4. MBNL1 Inhibits clonogenic capacity. 913 (A), 821 (B), 3691 (C), and 3832 (D) Neurospheres were electroporated with pLenti7.3-emGFP (GFP) or pLenti6-MBNL1 and challenged in a clonogenic assay in methylcellulose. Neurosphere diameter is presented as Poisson Distributions. Triplicate samples were used in these experiments. Statistics: student t test *** p<0.0001.

Figure 5. MBNL1 Inhibits Orthotopic GBM Xenograft Growth. (A) MBNL1 expression in pLenti6- empty and pLenti6-MBNL1 transduced 913 Luciferase neurospheres. (B) Tumor growth as measured by bioluminescence (Flux) in NSG mice bearing pLenti6 or pLenti6-MBNL1 transduced 913 orthotopic xenografts. (C) Representative mice from the study shown in B. (D) Survival analysis of mice from the study shown in B. (E) Quantitative Real-Time PCR analysis for MBNL1 mRNA expression in pLV-TRE- MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and treated with indicated concentrations of doxycycline. (F) Representative histogram depicting flow cytometric evaluation of mCherry positive cells in pLV-TRE-MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and treated with vehicle or 1500 nM doxycycline. (G) Averaged percentage of mCherry positive cells (left graph) and averaged expression intensity (fluorescent signal) of triplicates from F. (H) MBNL1 target gene splicing in pLV-TRE-MBNL1-mCherry transduced 913 neurospheres treated with vehicle or 1500 nM doxycycline. (I) Survival analysis of NSG mice bearing pLV-TRE-MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and fed standard, control, chow (black boxes) or chow containing 200mg/kg doxycycline (Bioserve) (red circles). Statistics: Student t-test *** p<0.001, **** p<0.0001; One-way ANOVA **** p<0.0001; Log-rank survival analysis ** p<0.01, *** p<0.001.

The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness

Dillon M. Voss, Anthony Sloan, Raffaella Spina, et al.

Cancer Res Published OnlineFirst September 14, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-1233

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/09/12/0008-5472.CAN-20-1233.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2020/09/12/0008-5472.CAN-20-1233. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.