Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

A phase 1b study of zilovertamab in combination with paclitaxel for locally advanced/unresectable or metastatic Her2-negative breast cancer.

BACKGROUND: Zilovertamab is a humanized monoclonal antibody targeting ROR1, an onco-embryonic antigen expressed by malignant cells of a variety of solid tumors, including breast cancer. A prior phase 1 study showed that zilovertamab was well tolerated and effective in inhibiting ROR1-signaling, which leads to activation of ERK1/2, NF-κB, and NRF2 target genes. This phase 1b study evaluated the safety and tolerability of zilovertamab with paclitaxel in patients with advanced breast cancer. PATIENTS AND METHODS: Eligible patients had locally advanced, unresectable, or metastatic HER2- breast cancer with Eastern Cooperative Group performance status of 0-2 and without prior taxane therapy in the advanced setting. Study treatment included 600 mg of zilovertamab administered intravenously (IV) on Days 1 and 15 of Cycle 1 and then Day 1 of each 28-day cycle along with paclitaxel weekly at 80 mg/m2 IV. RESULTS: Study patients had received a median of 4 prior therapies (endocrine therapy + chemotherapy) for locally advanced, unresectable, or metastatic disease. No patient discontinued therapy due to toxicity ascribed to zilovertamab. Adverse events were consistent with the known safety profile of paclitaxel. Of 16 patients, 6 (38%) had a partial response, and 6/16 (38%) patients had stable disease as best tumor response. CONCLUSION: The combination of zilovertamab and paclitaxel was safe and well tolerated in heavily pre-treated advanced breast cancer patients. Further evaluation of ROR1 targeting in breast cancer patients with zilovertamab is warranted. TRIAL REGISTRATION: NCT02776917. Registered on ClinicalTrials.gov on 05/17/2016.

Association of baseline ROR1 and ROR2 gene expression with clinical outcomes in the I-SPY2 neoadjuvant breast cancer trial.

PURPOSE: ROR1 and ROR2 are Type 1 tyrosine kinase-like orphan receptors for Wnt5a that are associated with breast cancer progression. Experimental agents targeting ROR1 and ROR2 are in clinical trials. This study evaluated whether expression levels of ROR1 or ROR2 correlated with one another or with clinical outcomes. METHODS: We interrogated the clinical significance of high-level gene expression of ROR1 and/or ROR2 in the annotated transcriptome dataset from 989 patients with high-risk early breast cancer enrolled in one of nine completed/graduated/experimental and control arms in the neoadjuvant I-SPY2 clinical trial (NCT01042379). RESULTS: High ROR1 or high ROR2 was associated with breast cancer subtypes. High ROR1 was more prevalent among hormone receptor-negative and human epidermal growth factor receptor 2-negative (HR-HER2-) tumors and high ROR2 was less prevalent in this subtype. Although not associated with pathologic complete response, high ROR1 or high ROR2 each was associated with event-free survival (EFS) in distinct subtypes. High ROR1 associated with a worse EFS in HR + HER2- patients with high post-treatment residual cancer burden (RCB-II/III) (HR 1.41, 95% CI = 1.11-1.80) but not in patients with minimal post-treatment disease (RCB-0/I) (HR 1.85, 95% CI = 0.74-4.61). High ROR2 associated with an increased risk of relapse in patients with HER2 + disease and RCB-0/I (HR 3.46, 95% CI = 1.33-9.020) but not RCB-II/III (HR 1.07, 95% CI = 0.69-1.64). CONCLUSION: High ROR1 or high ROR2 distinctly identified subsets of breast cancer patients with adverse outcomes. Further studies are warranted to determine if high ROR1 or high ROR2 may identify high-risk populations for studies of targeted therapies.

Impact of the Types and Relative Quantities of IGHV Gene Mutations in Predicting Prognosis of Patients With Chronic Lymphocytic Leukemia.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, "(S+Rc) to Rnc IGHV mutation ratio". When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

High expression level of ROR1 and ROR1-signaling associates with venetoclax resistance in chronic lymphocytic leukemia.

Although the BH3-mimetic venetoclax is highly cytotoxic for chronic lymphocytic leukemia (CLL) cells, some patients with CLL fail to clear minimal residual disease (MRD). We examined the CLL cells of seven such patients (CLL1-7) and found each had high-level expression of ROR1. By examining the CLL cells from such patients prior to therapy at SC1 and then more than 1 year later (Sample Collection 2 (SC2)), when they had progressive increases in MRD despite continued venetoclax therapy, we found the levels of ROR1 expressed on CLL cells at SC2 were significantly higher than that on CLL cells collected at SC1. At SC2, we also observed upregulation of genes induced by Wnt5a-induced ROR1 signaling, including BCL2L1. Transduction of the CLL-cell-line MEC1 to express ROR1 enhanced expression of target genes induced by ROR1-signaling, increased expression of BCL-XL, and enhanced resistance to venetoclax, even in MEC1 made to express mutant forms of BCL2, which are associated with venetoclax resistance. Treatment of primary CLL cells with Wnt5a also increased their resistance to venetoclax, an effect that could be inhibited by the anti-ROR1 mAb (UC-961, zilovertamab). Collectively, these studies indicate that Wnt5a-induced ROR1-signaling can enhance resistance to venetoclax therapy.

Wnt5a enhances proliferation of chronic lymphocytic leukemia and ERK1/2 phosphorylation via a ROR1/DOCK2-dependent mechanism.

Patients with chronic lymphocytic leukemia (CLL) have high plasma-levels of Wnt5a, which can induce phosphorylation of ERK1/2 and enhance CLL-cell proliferation. Such effects could be inhibited by treatment with an ERK1/2 inhibitor, ERK1/2-specific siRNA, or cirmtuzumab, an anti-ROR1 mAb. The CLL-derived line, MEC1, expresses Wnt5a, but not ROR1. MEC1 cells transfected to express ROR1 (MEC1-ROR1) had higher levels of phosphorylated ERK1/2 than parental MEC1, or MEC1 transfected with ROR1ΔPRD, a truncated ROR1 lacking the cytoplasmic proline-rich domain (PRD), or ROR1P808A a mutant ROR1 with a P→A substitution at 808, which is required for complexing with the Rac-specific-guanine-nucleotide-exchange factor DOCK2 upon stimulation with Wnt5a. We silenced DOCK2 with siRNA and found this repressed the capacity of Wnt5a to induce ERK1/2 phosphorylation in MEC1-ROR1 or CLL cells. CLL cells that expressed ROR1 had higher levels of phosphorylated ERK1/2 or DOCK2 than CLL cells lacking ROR1. Although we found ibrutinib could inhibit the phosphorylation of ERK1/2 and DOCK2 induced by B-cell-receptor ligation, we found that this drug was unable to inhibit Wnt5a-induced, ROR1-dependent phosphorylation of ERK1/2 or DOCK2. This study demonstrates that Wnt5a can induce activation of ERK1/2 and enhance CLL-cell proliferation via a ROR1/DOCK2-dependent pathway independent of BTK.

NF-κB-p62-NRF2 survival signaling is associated with high ROR1 expression in chronic lymphocytic leukemia.

Progression of chronic lymphocytic leukemia (CLL) and resistance to therapy are affected by tumor microenvironmental factors. One such factor is B-cell activating factor (BAFF), a cytokine that is produced mainly by nurse-like cells (NLC) and enhances CLL cells survival and modulates response to therapy. In CLL cells, BAFF activates NF-κB signaling, but how NF-κB supports CLL survival is not entirely clear. In this study we show that BAFF induces accumulation of the signaling and autophagy adaptor p62/SQSTM1 in a manner dependent on NF-κB activation. p62 potentiates mTORC1 signaling and activates NRF2, the master regulator of the anti-oxidant response. We found that expression of NRF2 target genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1), is particularly enriched in CLL cells with high ROR1 surface expression (ROR1Hi). ROR1Hi CLL cells with elevated NQO1 expression exhibit resistance to drugs that induce ROS accumulation, such venetoclax. However, such cells are more sensitive to compound 29h, a pro-drug that only becomes active after being metabolized by NQO1. Accordingly, 29h sensitizes high NQO1 CLL cells to venetoclax. Collectively, our study unravels a previously unknown signaling network through which the NF-κB-p62-NRF2 axis protects ROR1-high CLL cells from ROS-inducing therapeutics.

Cirmtuzumab blocks Wnt5a/ROR1 stimulation of NF-κB to repress autocrine STAT3 activation in chronic lymphocytic leukemia.

Coculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-κB within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-κB target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-κB. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-κB and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-κB and STAT3 in patients with CLL.

Activation of hedgehog signaling associates with early disease progression in chronic lymphocytic leukemia.

Targeted sequencing of 103 leukemia-associated genes in leukemia cells from 841 treatment-naive patients with chronic lymphocytic leukemia (CLL) identified 89 (11%) patients as having CLL cells with mutations in genes encoding proteins that putatively are involved in hedgehog (Hh) signaling. Consistent with this finding, there was a significant association between the presence of these mutations and the expression of GLI1 (χ2 test, P < .0001), reflecting activation of the Hh pathway. However, we discovered that 38% of cases without identified mutations also were GLI1+ Patients with GLI1+ CLL cells had a shorter median treatment-free survival than patients with CLL cells lacking expression of GLI1 independent of IGHV mutation status. We found that GANT61, a small molecule that can inhibit GLI1, was highly cytotoxic for GLI1+ CLL cells relative to that of CLL cells without GLI1. Collectively, this study shows that a large proportion of patients have CLL cells with activated Hh signaling, which is associated with early disease progression and enhanced sensitivity to inhibition of GLI1.

A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion.

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.

Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody.

Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.

Splicing modulation sensitizes chronic lymphocytic leukemia cells to venetoclax by remodeling mitochondrial apoptotic dependencies.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eµ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.

Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia.

Cirmtuzumab is a humanized monoclonal antibody (mAb) that targets ROR1, an oncoembryonic orphan receptor for Wnt5a found on cancer stem cells (CSCs). Aberrant expression of ROR1 is seen in many malignancies and has been linked to Rho-GTPase activation and cancer stem cell self-renewal. For patients with chronic lymphocytic leukemia (CLL), self-renewing, neoplastic B cells express ROR1 in 95% of cases. High-level leukemia cell expression of ROR1 is associated with an unfavorable prognosis. We conducted a phase 1 study involving 26 patients with progressive, relapsed, or refractory CLL. Patients received four biweekly infusions, with doses ranging from 0.015 to 20 mg/kg. Cirmtuzumab had a long plasma half-life and did not have dose-limiting toxicity. Inhibition of ROR1 signaling was observed, including decreased activation of RhoA and HS1. Transcriptome analyses showed that therapy inhibited CLL stemness gene expression signatures in vivo. Cirmtuzumab is safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL.

MicroRNA dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia.

Loss of miR-15/16 is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of BCL2, which factors in leukemia pathogenesis. Indeed, an inhibitor of Bcl2, venetoclcax, is highly active in the treatment of patients with CLL. However, single-agent venetoclcax fails to eradicate minimal residual disease in most patients. Accordingly, we were interested in other genes that may be regulated by miR-15/16, which may target other drivers in CLL. We found that miR-15/16 targets ROR1, which encodes an onco-embryonic surface protein expressed on the CLL cells of over 90% of patients, but not on virtually all normal postpartum tissues. CLL with high-level expression of ROR1 also have high-level expression of Bcl2, but low-to-negligible miR-15/16 Moreover, CLL cases with high-level ROR1 have deletion(s) at the chromosomal location of the genes encoding miR-15/16 (13q14) more frequently than cases with low-to-negligible ROR1, implying that deletion of miR-15/16 may promote overexpression of ROR1, in addition to BCL2 ROR1 is a receptor for Wnt5a, which can promote leukemia-cell proliferation and survival, and can be targeted by cirmtuzumab, a humanized anti-ROR1 mAb. We find that this mAb can enhance the in vitro cytotoxic activity of venetoclcax for CLL cells with high-level expression of ROR1, indicating that combining these agents, which target ROR1 and Bcl2, may have additive, if not synergistic, activity in patients with this disease.

Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia.

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.

High-level ROR1 associates with accelerated disease progression in chronic lymphocytic leukemia.

ROR1 is an oncoembryonic orphan receptor found on chronic lymphocytic leukemia (CLL) B cells, but not on normal postpartum tissues. ROR1 is a receptor for Wnt5a that may complex with TCL1, a coactivator of AKT that is able to promote development of CLL. We found the CLL cells of a few patients expressed negligible ROR1 (ROR1Neg), but expressed TCL1A at levels comparable to those of samples that expressed ROR1 (ROR1Pos). Transcriptome analyses revealed that ROR1Neg cases generally could be distinguished from those that were ROR1Pos in unsupervised gene-expression clustering analysis. Gene-set enrichment analyses demonstrated that ROR1Neg CLL had lower expression and activation of AKT signaling pathways relative to ROR1Pos CLL, similar to what was noted for leukemia that respectively developed in TCL1 vs ROR1xTCL1 transgenic mice. In contrast to its effect on ROR1Pos CLL, Wnt5a did not enhance the proliferation, chemotaxis, or survival of ROR1Neg CLL. We examined the CLL cells from 1568 patients, which we randomly assigned to a training or validation set of 797 or 771 cases, respectively. Using recursive partitioning, we defined a threshold for ROR1 surface expression that could segregate samples of the training set into ROR1-Hi vs ROR1-Lo subgroups that differed significantly in their median treatment-free survival (TFS). Using this threshold, we found that ROR1-Hi cases had a significantly shorter median TFS and overall survival than ROR1-Lo cases in the validation set. These data demonstrate that expression of ROR1 may promote leukemia-cell activation and survival and enhance disease progression in patients with CLL.

Dielectrophoretic recovery of DNA from plasma for the identification of chronic lymphocytic leukemia point mutations.

AIM: Circulating cell free (ccf) DNA contains information about mutations affecting chronic lymphocytic leukemia (CLL). The complexity of isolating DNA from plasma inhibits the development of point-of-care diagnostics. Here, we introduce an electrokinetic method that enables rapid recovery of DNA from plasma. MATERIALS & METHODS: ccf-DNA was isolated from 25 µl of CLL plasma using dielectrophoresis. The DNA was used for PCR amplification, sequencing and analysis. RESULTS: The ccf-DNA collected from plasma of 5 CLL patients revealed identical mutations to those previously identified by extracting DNA from CLL cells from the same patients. CONCLUSION: Rapid dielectrophoresis isolation of ccf-DNA directly from plasma provides sufficient amounts of DNA to use for identification of point mutations in genes associated with CLL progression.

Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide-B.

RNA splicing plays a fundamental role in human biology. Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematologic malignancies. We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from patients with chronic lymphocytic leukemia and leukemia-lymphoma cell lines. We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention - spliceosome modulation. This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes. The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment. In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model. Overall, these findings give evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide an additional rationale for the development of spliceosome modulators for cancer therapy.

Transcriptome sequencing reveals potential mechanism of cryptic 3' splice site selection in SF3B1-mutated cancers.

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.