Lindsay D. Smith

Design principles for bifunctional targeted oligonucleotide enhancers of splicing

Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a ‘tail’ of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns.

The PI3K/mTOR inhibitor PF-04691502 induces apoptosis and inhibits microenvironmental signaling in CLL and the Eµ-TCL1 mouse model

Current treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti-immunoglobulin M-induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eμ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

BRCA1 exon 11 a model of long exon splicing regulation

BRCA1 exon 11 is one of the biggest human exons, spanning 3426 bases. This gene is potentially involved in DNA repair as well as cell growth and cell cycle control. Exon 11 is regulated at the splicing level producing three main different combinations of BRCA1 mature transcripts; one including the whole of exon 11 (full isoform), one skipping the entire exon (D11 isoform), and one including only 117 base pairs of exon 11 (D11q isoform). Using minigene and deletion analyses, we have previously described important splicing regulatory sequences located at the beginning of this exon (5′ end). We have now found additional important sequences located at its 3′ end. In particular, we describe the presence of a strong splicing enhancer adjacent to the downstream 5′ splice site, which minimizes competition from an upstream 5′ splice site and so ensures long exon inclusion. Analyses of the proteins binding these RNA sequences have revealed that Tra2beta and hnRNP L are involved in the regulation of BRCA1 exon 11 by influencing the recognition of donor sites. Interestingly, BRCA1 exon 11 carrying deletion of the regulatory sequences bound by these factors also showed unexpected responses to up- or downregulation of these regulatory proteins, suggesting that they can also bind elsewhere in this large exon and elicit different effects on its recognition. The identification of sequences and proteins relevant for the regulation of BRCA1 exon 11 now provides better knowledge on how this exon is recognized and may represent an important step toward understanding how large exons are regulated.

The dual Syk/JAK inhibitor cerdulatinib antagonizes B-cell receptor and microenvironmental signaling in chronic lymphocytic leukemia

Purpose: B-cell receptor (BCR)–associated kinase inhibitors, such as ibrutinib, have revolutionized the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative, and resistance is already emerging in a proportion of patients. IL4, expressed in CLL lymph nodes, can augment BCR signaling and reduce the effectiveness of BCR kinase inhibitors. Therefore, simultaneous targeting of the IL4- and BCR signaling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients. Experimental Design: PBMCs from patients with CLL were treated in vitro with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine, and cell signaling assay were performed and analyzed by flow cytometry, immunoblotting, q-PCR, and ELISA as indicated. Results: At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL4-induced downstream signaling in CLL cells using multiple readouts and prevented anti-IgM- and nurse-like cell (NLC)–mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV-unmutated samples with greater BCR signaling capacity and response to IL4, or samples expressing higher levels of sIgM, CD49d+, or ZAP70+. Cerdulatinib overcame anti-IgM, IL4/CD40L, or NLC-mediated protection by preventing upregulation of MCL-1 and BCL-XL; however, BCL-2 expression was unaffected. Furthermore, in samples treated with IL4/CD40L, cerdulatinib synergized with venetoclax in vitro to induce greater apoptosis than either drug alone. Conclusions: Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. Clin Cancer Res; 23(9); 2313–24. ©2016 AACR.

A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

Summary The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.

Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3’ Splice Site Recognition

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.

Novel splice‐switching oligonucleotide promotes BRCA1 aberrant splicing and susceptibility to PARP inhibitor action

Tumors carrying hereditary mutations in BRCA1, which attenuate the BRCA1 DNA damage repair pathway, are more susceptible to dual treatment with PARP inhibitors and DNA damaging therapeutics. Conversely, breast cancer tumors with nonmutated functional BRCA1 are less sensitive to PARP inhibition. We describe a method that triggers susceptibility to PARP inhibition in BRCA1‐functional tumor cells. BRCA1 exon 11 is a key for the function of BRCA1 in DNA damage repair. Analysis of the BRCA1 exon 11 splicing mechanism identified a key region within this exon which, when deleted, induced exon 11 skipping. An RNA splice‐switching oligonucleotide (SSO) developed to target this region was shown to artificially stimulate skipping of exon 11 in endogenous BRCA1 pre‐mRNA. SSO transfection rendered wild‐type BRCA1 expressing cell lines more susceptible to PARP inhibitor treatment, as demonstrated by a reduction in cell survival at all SSO concentrations tested. Combined SSO and PARP inhibitor treatment increased γH2AX expression indicating that SSO‐dependent skipping of BRCA1 exon 11 was able to promote DSBs and therefore synthetic lethality. In conclusion, this SSO provides a new potential therapeutic strategy for targeting BRCA1‐functional breast cancer by enhancing the effect of PARP inhibitors.

Abstract 1871: Development of pelorol analogues to activate the SHIP1 lipid phosphatase; a novel paradigm to suppress B-cell receptor signaling in human B-cell cancers

Signaling via the B-cell receptor (BCR) is a major driver of malignant B-cell proliferation and survival in B-cell malignancies including chronic lymphocytic leukemia (CLL). The role of kinases in BCR signalling is well understood and kinase inhibitors are effective therapies for B-cell cancers. However, resistance is increasingly common and new drugs are required. In this study we investigated an alternate strategy to block BCR signaling via small molecule activation of SHIP1, an inositol lipid phosphatase which suppresses PI3 kinase (PI3K)-mediated signaling by catalysing the conversion of the PI3K product PI(3,4,5)P3 (PIP3) to PI(3,4)P2. We focused on the exemplar compound, AQX-C5, which is structurally related to the natural product pelorol. AQX-C5 interfered with the ability of anti-IgM to activate PIP3-dependent signaling, including downstream phosphorylation of AKT, ERK1/2 and p70-S6K, and induction of MYC, in primary CLL cells. AQX-C5 also induced CLL cell apoptosis and overcame the survival-promoting effects of anti-IgM or CD40-ligand/interleukin-4. CLL cells were more sensitive to pro-apoptotic effects of AQX-C5 compared to non-malignant B cells. The AQX-C5-induced apoptosis in CLL cells was associated with down-modulation of the BCL2-related survival protein MCL1 in anti-IgM-stimulated cells, and induction of the pro-apoptotic protein NOXA. In addition to effects on PIP3-dependent signaling, AQX-C5 triggered down-modulation of CXCR4, a chemokine receptor important for homing of malignant cells to supportive tissue microenvironments. CXCR4 downmodulation was selective, since AQX-C5 did not alter surface expression of other receptors, including IgM and the transferrin receptor. In contrast to AQX-C5, the PI3Kδ inhibitor idelalisib failed to alter CXCR4 expression, indicating that these effects of AQX-C5 may be mediated via PI(3,4)P2 accumulation rather than PIP3 depletion. We evaluated the in vivo anti-lymphoma activity of AQX-C5 in a xenograft model using the TMD8 cell line, derived from a diffuse large B-cell lymphoma. Similar to the BTK inhibitor ibrutinib, AQX-C5 substantially reduced the rate of tumor growth and tumor mass at the end of the experiment, without evidence for gross toxicity. These findings suggest that SHIP1 activators, such as AQX-C5, may be interesting therapeutic agents for various B-cell cancers. Citation Format: Graham Packham, Elizabeth Lemm, Beatriz Valle-Argos, Lindsay Smith, Nichola Weston-Bell, Freda Stevenson, Matthew J. Carter, Mark S. Cragg, Francesco Forconi, Andrew J. Steele, Jennifer Cross, Curtis Harwig, Georg Lenz, Lloyd Mackenzie, Pavel Klener. Development of pelorol analogues to activate the SHIP1 lipid phosphatase; a novel paradigm to suppress B-cell receptor signaling in human B-cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1871.

A study of splicing mutations in disorders of sex development.

The presence of splicing sequence variants in genes responsible for sex development in humans may compromise correct biosynthesis of proteins involved in the normal development of gonads and external genitalia. In a cohort of Brazilian patients, we identified mutations in HSD17B3 and SRD5A2 which are both required for human sexual differentiation. A number of these mutations occurred within regions potentially critical for splicing regulation. Minigenes were used to validate the functional effect of mutations in both genes. We evaluated the c.277 + 2 T > G mutation in HSD17B3, and the c.544 G > A, c.548-44 T > G and c.278delG mutations in SRD5A2. We demonstrated that these mutations altered the splicing pattern of these genes. In a genomic era these results illustrate, and remind us, that sequence variants within exon-intron boundaries, which are primarily identified for diagnostic purposes and have unknown pathogenicity, need to be assessed with regards to their impact not only on protein expression, but also on mRNA splicing.