
==== Front
J Mol Cell Biol
J Mol Cell Biol
jmcb
Journal of Molecular Cell Biology
1674-2788
1759-4685
Oxford University Press

37827547
10.1093/jmcb/mjad063
mjad063
Perspective
AcademicSubjects/SCI01180
Targeting dysregulated splicing factors in cancer: lessons learned from RBM10 deficiency
https://orcid.org/0000-0003-4383-9423
Wang Yongbo Minhang Hospital, Department of Cellular and Genetic Medicine, School of Basic Medical Sciences, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China

https://orcid.org/0000-0002-6605-3637
Wang Zefeng CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China

Correspondence to: Yongbo Wang, E-mail: wangyongbo@fudan.edu.cn
Correspondence to: Zefeng Wang, E-mail: wangzefeng@picb.ac.cn
10 2023
12 10 2023
12 10 2023
15 10 mjad06318 7 2023
26 9 2023
11 10 2023
04 4 2024
© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.
2023
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
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pmcRibonucleic acid (RNA) splicing dysregulation is considered a molecular hallmark and important therapeutic target of cancer. Although targeting splicing has been intensively pursued for cancer therapy, specific modulators of dysregulated splicing factors are generally lacking. RNA binding motif protein 10 (RBM10) is frequently mutated in multiple cancers, in particular lung adenocarcinoma (LUAD). Increasing evidence has demonstrated that RBM10 deficiency due to loss-of-function (LOF) mutation or aberrant expression contributes to cancer development, progression, and therapeutic response. Here, we summarize the functional consequences and new therapeutic implications of RBM10 deficiency in cancer. Using RBM10 as a representative example, we highlight main strategies for targeting dysregulated splicing factors. These strategies, either alone or in combination with currently approved therapies, may hold great promise in cancer treatment.

Introduction

Precursor RNA (pre-RNA) splicing, i.e. the excision of non-coding introns and ligation of exons catalyzed by the spliceosome, is a fundamental step in human gene expression. RNA splicing is highly regulated, as almost all human genes are subject to alternative splicing (AS). AS enables the generation of multiple RNA isoforms from a single gene, greatly expanding the functional diversity of the human transcriptome (Rogalska et al., 2023). Hundreds of trans-acting factors (i.e. splicing factors) participate in AS regulation, primarily through specifically recognizing regulatory cis-elements in the pre-RNA to promote or suppress splicing reactions (Matera and Wang, 2014). AS regulation plays crucial roles in many biological processes, whereas its dysregulation is a major cause of human diseases, including cancer (Bradley and Anczuków, 2023; Rogalska et al., 2023).

Splicing dysregulation is pervasive in cancer, mainly arising from the mutation or aberrant expression of splicing factors, as well as mutations in splicing regulatory cis-elements. The most studied examples of cancer-related splicing dysregulation are recurrent mutations of the splicing factors SF3B1, U2AF1, and SRSF2 in hematopoietic malignancies as well as solid tumors (Bradley and Anczuków, 2023). In addition, a growing number of dysregulated splicing factors (e.g. RBM10 and SRSF1) and splicing events (e.g. MET exon 14 skipping and anti-apoptotic Bcl-xL isoform) have been shown to play critical roles in cancer pathogenesis and therapy (Bradley and Anczuków, 2023). Therefore, splicing dysregulation is regarded as a new molecular hallmark of cancer and has been intensively pursued as a therapeutic target (Wang et al., 2020; Bradley and Anczuków, 2023). Although exciting progress has been made in targeting splicing alterations in cancer, strategies for targeting splicing factors, particularly those functioning as tumor suppressors, are still lacking.

RBM10 alterations and functions in cancer

RBM10 is an auxiliary component of the spliceosome that regulates AS by binding to specific regions of cognate pre-mRNAs (Bechara et al., 2013; Wang et al., 2013). It has been implicated in a complex splicing regulatory network. On the one hand, RBM10 was reported to interact with multiple other splicing factors (Hegele et al., 2012). On the other hand, it regulates the AS of various splicing factors, including its closest paralogue RBM5 (Sun et al., 2017). Notably, non-splicing functions of RBM10, such as chromatin remodeling (Weigt et al., 2021) and RNA 3′ untranslated region (3′UTR) processing (Mohan et al., 2018), have also been proposed. However, the exact mechanisms by which RBM10 exerts splicing and non-splicing functions have not been fully elucidated.

Germline LOF mutations in RBM10 cause a severe congenital developmental disorder known as TARP syndrome (MIM number 311900). Somatic mutations in RBM10 frequently occur in several cancers, as summarized in Bao et al. (2023), including LUAD, bladder carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma. It remains an intriguing question why germline mutations in RBM10 lead to developmental defects in multiple organs, while somatic mutations occur in various types of cancers. We speculate that RBM10 mutations may affect different target genes in distinct cell types or developmental stages, thereby leading to disparate phenotypes. To date, the functional consequences of RBM10 mutations in cancer have been mostly investigated in LUAD.

In LUAD, RBM10 mutations are primarily LOF mutaions and often co-occur with epidermal growth factor receptor (EGFR) or KRAS driver mutations (Zhao et al., 2017; Bao et al., 2023). Because RBM10 is located on the X-chromosome, RBM10 loss could be caused by hemizygous mutation in males or heterozygous mutation of the active allele in females via X-chromosome inactivation. This is different from cancer-associated heterozygous mutations in SF3B1, U2AF1, and SRSF2, which often lead to changes in normal functions (Bradley and Anczuków, 2023). RBM10 has been reported to be a tumor suppressor in LUAD, and loss of RBM10 promotes LUAD development, progression, and therapeutic response (Rogers et al., 2018; Bao et al., 2023). RBM10 suppresses LUAD by regulating the AS of key target genes, such as NUMB (Bechara et al., 2013) and eukaryotic translation initiation factor 4H (EIF4H) (Zhang et al., 2020; Figure 1A). Specifically, we showed that RBM10 inhibits the inclusion of exon 5 in EIF4H, which plays an essential role in LUAD cell proliferation and survival (Zhang et al., 2020). In addition, RBM10 deficiency was shown to compromise the efficacy of EGFR tyrosine kinase inhibitor (TKI), partially by regulating the AS of the anti-apoptotic gene Bcl-x (Nanjo et al., 2022; Figure 1A). RBM10 deficiency is also correlated with key regulators of the immune microenvironment in LUAD (Liu et al., 2021) and thus may affect the response to immunotherapy. Notably, mechanisms other than AS regulation were proposed for the tumor-suppressive activities of RBM10 (Cao et al., 2021). A few studies also reported the oncogenic activities of RBM10 (Loiselle and Sutherland, 2018), suggesting distinct RBM10 functions in different physiopathological contexts.

Figure 1 Strategies for targeting RBM10 deficiency in cancer. (A) Targeting oncogenic splicing isoforms of key target genes of RBM10. Splicing isoforms enhanced by RBM10 deficiency can be specifically blocked by splicing-switching ASOs, as exemplified by EIF4H exon 5 inclusion in LUAD. (B) Targeting vulnerabilities resulting from RBM10 deficiency. RBM10 deficiency sensitized cancer cells to anti-cancer agents, such as spliceosome or Wee1 kinase inhibitor in LUAD, venetoclax in AML, and anti-PD1 antibody in HCC. (C) Restoration of RBM10 expression. AAV- or mRNA-based therapies may be used to restore RBM10 expression in cancers, such as LUAD, where RBM10 acts as a tumor suppressor. (D) Combining splicing modulators with currently existing therapeutic approaches. Combination therapy can improve the efficacy and overcome the resistance to conventional treatment approaches in cancers with RBM10 deficiency.

In addition, genetic ablation of RBM10 was reported to significantly augment the response to venetoclax-based chemotherapy in acute myeloid leukemia (AML) (Figure 1B). This effect is in part attributed to mis-splicing of X-linked inhibitor of apoptosis (XIAP) (Wang et al., 2023), which highlights the promise of depleting RBM10 to enhance the efficacy of chemotherapy in treating AML. Another intriguing study revealed that Schlafen 11 (SLFN11) deficiency induced CCL2 signaling and macrophage M2 polarization and thus potentiated the efficacy of anti-PD1 therapy in treating hepatocellular carcinoma (HCC) (Figure 1B). SLFN11 and the E3 ligase tripartite motif containing 21 (TRIM21) competitively bind to RBM10, and SLFN11 deficiency enhances TRIM21-mediated RBM10 degradation and consequent NUMB exon 9 inclusion (Zhou et al., 2023). This study suggested that RBM10 deficiency may rewire the tumor microenvironment, thereby enhancing the efficacy of immune checkpoint inhibitors (ICIs) in treating HCC. In addition, RBM10 was shown to physically interact with the tumor suppressor p53 in colon cancer, which disrupted the MDM2–p53 interaction and subsequently stabilized p53 to suppress tumor development (Jung et al., 2020). Furthermore, RBM10 was found to be associated with shorter progression-free survival in metastatic colorectal cancer (CRC) patients, while it was suggested to be a tumor suppressor in CRC development (Guo et al., 2023). Together, these studies indicate that RBM10 has important and probably context-dependent functions in cancer.

Targeting dysregulated splicing factors in cancer: insights from RBM10 deficiency

General approaches for splicing modulation in cancer include targeting spliceosomal components, splicing factors, and individual splicing isoforms. Spliceosome inhibitors such as E7107 exhibited potent anti-cancer effects in pre-clinical studies, but further drug development was suspended due to the observed toxicity in clinical trials (Effenberger et al., 2017). This prompted the development of specific modulators for splicing factors with high efficacy and low toxicity. Focusing on RBM10 deficiency, we summarize main strategies for targeting dysregulated splicing factors in cancer.

The first strategy is to modulate the AS of key target genes of RBM10 (Figure 1A). For example, the inclusion of EIF4H exon 5 is significantly upregulated by RBM10 loss, and antisense oligonucleotide (ASO) was used to effectively inhibit this AS event and suppress LUAD development (Zhang et al., 2020). In addition, the anti-apoptotic Bcl-xL isoform level is elevated by RBM10 deficiency, limiting the response to the third-generation EGFR TKI osimertinib in EGFR-mutant LUAD. Thus, a Bcl-xL inhibitor was used together with osimertinib to synergistically inhibit LUAD (Nanjo et al., 2022).

The second strategy is to target vulnerabilities resulting from RBM10 deficiency (Figure 1B). For instance, RBM10 loss increased the sensitivity of EGFR-mutant LUAD to spliceosome inhibitors (Bao et al., 2023; Figure 1B). In addition, a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) screen identified synthetic lethal genes for RBM10 deficiency in LUAD, including Aurora A and WEE1 kinases (Machour et al., 2023; Figure 1B). RBM10-deficient LUAD cells are selectively sensitive to WEE1 inhibitors, which is attributed to DNA damage accumulation, replication stress, and premature mitotic entry, rather than splicing alterations (Machour et al., 2023). Interestingly, RBM10 was reported to positively regulate BRCA1 expression via AS coupled to nonsense-mediated mRNA decay and participate in the maintenance of genome stability (Nie et al., 2023). Accordingly, RBM10-deficient cells were shown to be sensitive to agents targeting BRCA1 mutations, such as poly(ADP-ribose) polymerase inhibitor (PARPi) (Nie et al., 2023). Furthermore, RBM10 depletion sensitized AML cells to the chemotherapeutic agent venetoclax partially through mis-splicing of XIAP (Wang et al., 2023; Figure 1B). High-throughput genetic and pharmacological screens will be powerful tools for identifying new genetic vulnerabilities as well as therapeutic compounds specific for RBM10 deficiency in cancer (Figure 1B).

The third strategy can be called gene restoration (Figure 1C). Adeno-associated virus (AAV)-based gene therapy has been shown to restore genes disrupted in genetic diseases and is being tested in cancer treatment (Li and Samulski, 2020). mRNA delivery has been successfully used for anti-viral vaccines and is investigated for developing immunogenic vaccines and tumor suppressor restoring agents (Qin et al., 2022). In addition, CRISPR/Cas systems have been modified to correct genetic mutations, which may be exploited to repair specific mutations and restore gene functions ex vivo or in vivo in cancer (Katti et al., 2022). Further studies are required to examine the feasibility of RBM10 restoration for treating RBM10-deficient cancers (Figure 1C).

The fourth strategy is to target the upstream regulators of RBM10, which unfortunately are still generally unclear. Theoretically, with increasing understanding of RBM10 regulation, the repressed wild-type RBM10 in cancers could be activated though modulating its regulators to impede tumor growth. This is analogous to the activation of p53 by targeting MDM2, a well-established upstream regulator of the tumor suppressor p53.

Finally, the combinations of splicing modulators with currently existing targeted therapy, chemotherapy, or immunotherapy represent promising strategies for combating cancer (Figure 1D). In RBM10-deficient LUAD, the combination of a Bcl-xL inhibitor or a spliceosome inhibitor with osimertinib drastically improved the efficacy and overcame the resistance to TKI (Nanjo et al., 2022; Bao et al., 2023; Figure 1D). In AML, the splicing-dependent kinase inhibitor synergized with venetoclax and overcame the resistance to venetoclax by modulating splicing factor phosphorylation and XIAP splicing (Wang et al., 2023; Figure 1D). Since RBM10 loss can sensitize AML cells to venetoclax, we envisage that RBM10 degraders, such as proteolysis targeting chimera (PROTAC) molecules, may improve the effectiveness of venetoclax in AML (Figure 1D).

The therapeutic strategies based on RBM10 deficiency in cancer should be readily extended to other splicing factors. For example, RBM4 was found to function as a tumor suppressor in several cancers in part by suppressing Bcl-xL, the anti-apoptotic isoform of BCL2L1 (Wang et al., 2014). This oncogenic splicing event can be inhibited by ASO to impede the growth of RBM4-downregulated tumors (Bradley and Anczuków, 2023). Moreover, through synthetic lethal pharmaceutical screens, a recent study revealed that cancer cells with SF3B1 mutations are selectively sensitive to PARPi due to a defective replication stress response (Bland et al., 2023).

Aside from these strategies, the oncogenic splicing factors can be directly targeted by inhibitors, degraders, or splicing factor decoys (Bashari et al., 2023). For instance, degradation of the splicing factor RBM39 by indisulam or inhibition of protein arginine methyltransferases by MS-023 significantly enhanced the sensitivity of cancer cells to ICIs across several cancer types (Lu et al., 2021). Because splicing factors have important biological functions, the potential toxicity of splicing factor modulators needs to be carefully evaluated. Other major challenges, including delivery and clinical efficacy, should also be tackled when translating splicing factor modulators from bench to bedside.

Conclusion

Targeting dysregulated splicing factors, either by itself or in combination with other therapies, holds great promise for cancer treatment. An increasing number of studies have demonstrated the critical roles of RBM10 deficiency in cancer pathogenesis and therapy. Based on the new lessons learned from RBM10, we summarize main strategies for targeting splicing factors that are dysregulated in cancer. These strategies are anticipated to be tested in the clinic in the coming years and eventually benefit cancer patients.

[Dr Yufang Bao from School of Basic Medical Sciences, Fudan University helped creating the figure with BioRender.com. We acknowledge studies that contribute to the topic but were not cited in this essay due to limitation in space. This work was supported by the National Natural Science Foundation of China (81871878, 32030064, and 31730110), Shanghai Municipal Natural Science Fund (20ZR1406500), the Innovation Research Team of High-level Local Universities in Shanghai, the Strategic Priority Research Program of Chinese Academy of Sciences (CAS) (XDB38040100), the National Key Research and Development Program of China (2021YFA1300503), and the Starry Night Science Fund at Shanghai Institute for Advanced Study of Zhejiang University (SN-ZJU-SIAS-009). Z.W. is also supported by CAS Interdisciplinary Innovation Team and the type A CAS Pioneer 100-Talent Program.]
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References

Bao  Y., Zhang  S., Zhang  X.  et al. (2023). RBM10 loss promotes EGFR-driven lung cancer and confers sensitivity to spliceosome inhibition. Cancer Res.  83 , 1490–1502.36853175
Bashari  A., Siegfried  Z., Karni  R. (2023). Targeting splicing factors for cancer therapy. RNA  29 , 506–515.36697261
Bechara  E.G., Sebestyén  E., Bernardis  I.  et al. (2013). RBM5, 6, and 10 differentially regulate NUMB alternative splicing to control cancer cell proliferation. Mol. Cell  52 , 720–733.24332178
Bland  P., Saville  H., Wai  P.T.  et al. (2023). SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response. Nat. Genet.  55 , 1311–1323.37524790
Bradley  R.K., Anczuków  O. (2023). RNA splicing dysregulation and the hallmarks of cancer. Nat. Rev. Cancer  23 , 135–155.36627445
Cao  Y., Di  X., Zhang  Q.  et al. (2021). RBM10 regulates tumor apoptosis, proliferation, and metastasis. Front. Oncol.  11 , 603932.33718153
Effenberger  K.A., Urabe  V.K., Jurica  M.S. (2017). Modulating splicing with small molecular inhibitors of the spliceosome. Wiley Interdiscip. Rev. RNA  8 , 10.1002/wrna.1381.
Guo  L., Wang  Y., Yang  W.  et al. (2023). Molecular profiling provides clinical insights into targeted and immunotherapies as well as colorectal cancer prognosis. Gastroenterology  165 , 414–428.37146911
Hegele  A., Kamburov  A., Grossmann  A.  et al. (2012). Dynamic protein–protein interaction wiring of the human spliceosome. Mol. Cell  45 , 567–580.22365833
Jung  J.H., Lee  H., Cao  B.  et al. (2020). RNA-binding motif protein 10 induces apoptosis and suppresses proliferation by activating p53. Oncogene  39 , 1031–1040.31591476
Katti  A., Diaz  B.J., Caragine  C.M.  et al. (2022). CRISPR in cancer biology and therapy. Nat. Rev. Cancer  22 , 259–279.35194172
Li  C., Samulski  R.J. (2020). Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet.  21 , 255–272.32042148
Liu  B., Wang  Y., Wang  H.  et al. (2021). RBM10 deficiency is associated with increased immune activity in lung adenocarcinoma. Front. Oncol.  11 , 677826.34367963
Loiselle  J.J., Sutherland  L.C. (2018). RBM10: harmful or helpful—many factors to consider. J. Cell. Biochem.  119 , 3809–3818.29274279
Lu  S.X., De Neef  E., Thomas  J.D.  et al. (2021). Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Cell  184 , 4032–4047.34171309
Machour  F.E., Abu-Zhayia  E., Kamar  J.  et al. (2023). Harnessing DNA replication stress to target RBM10 deficiency in lung adenocarcinoma. bioRxiv, 10.1101/2023.02.19.529108
Matera  A.G., Wang  Z. (2014). A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol.  15 , 108–121.24452469
Mohan  N., Kumar  V., Kandala  D.T.  et al. (2018). A splicing-independent function of RBM10 controls specific 3′UTR processing to regulate cardiac hypertrophy. Cell Rep.  24 , 3539–3553.30257214
Nanjo  S., Wu  W., Karachaliou  N.  et al. (2022). Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR-mutant lung cancer. J. Clin. Invest.  132 , e145099.35579943
Nie  C., Zhou  X.A., Zhou  J.  et al. (2023). A transcription-independent mechanism determines rapid periodic fluctuations of BRCA1 expression. EMBO J.  42 , e111951.37334492
Qin  S., Tang  X., Chen  Y.  et al. (2022). mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct. Target. Ther.  7 , 166.35597779
Rogalska  M.E., Vivori  C., Valcárcel  J. (2023). Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects. Nat. Rev. Genet.  24 , 251–269.36526860
Rogers  Z.N., McFarland  C.D., Winters  I.P.  et al. (2018). Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice. Nat. Genet.  50 , 483–486.29610476
Sun  Y., Bao  Y., Han  W.  et al. (2017). Autoregulation of RBM10 and cross-regulation of RBM10/RBM5 via alternative splicing-coupled nonsense-mediated decay. Nucleic Acids Res.  45 , 8524–8540.28586478
Wang  E., Pineda  J.M.B., Kim  W.J.  et al. (2023). Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia. Cancer Cell  41 , 164–180.36563682
Wang  Y., Bao  Y., Zhang  S.  et al. (2020). Splicing dysregulation in cancer: from mechanistic understanding to a new class of therapeutic targets. Sci. China Life Sci.  63 , 469–484.32086672
Wang  Y., Chen  D., Qian  H.  et al. (2014). The splicing factor RBM4 controls apoptosis, proliferation, and migration to suppress tumor progression. Cancer Cell  26 , 374–389.25203323
Wang  Y., Gogol-Döring  A., Hu  H.  et al. (2013). Integrative analysis revealed the molecular mechanism underlying RBM10-mediated splicing regulation. EMBO Mol. Med.  5 , 1431–1442.24000153
Weigt  M., Gao  Q., Ban  H.  et al. (2021). Rbm10 facilitates heterochromatin assembly via the Clr6 HDAC complex. Epigenetics Chromatin  14 , 8.33468217
Zhang  S., Bao  Y., Shen  X.  et al. (2020). RNA binding motif protein 10 suppresses lung cancer progression by controlling alternative splicing of eukaryotic translation initiation factor 4H. EBioMedicine  61 , 103067.33130397
Zhao  J., Sun  Y., Huang  Y.  et al. (2017). Functional analysis reveals that RBM10 mutations contribute to lung adenocarcinoma pathogenesis by deregulating splicing. Sci. Rep.  7 , 40488.28091594
Zhou  C., Weng  J., Liu  C.  et al. (2023). Disruption of SLFN11 deficiency-induced CCL2 signaling and macrophage M2 polarization potentiates anti-PD-1 therapy efficacy in hepatocellular carcinoma. Gastroenterology  164 , 1261–1278.36863689
