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 ( = 98 CLL patients) and healthy volunteers ( = 9).

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.

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.

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.

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.

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.

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.

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.

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 (χ test, < .0001), reflecting activation of the Hh pathway.

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.

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).

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.

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.

dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia.

Loss of is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of , 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.

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.

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 (ROR1), but expressed TCL1A at levels comparable to those of samples that expressed ROR1 (ROR1).

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.

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.

Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia.

Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (>1 × 10(12)) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition.

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.

Ovarian cancer stem cells express ROR1, which can be targeted for anti-cancer-stem-cell therapy.

Although initially responsive to chemotherapy, many patients with ovarian cancer subsequently develop relapsed and potentially fatal metastatic disease, which is thought to develop from cancer stem cells (CSCs) that are relatively resistant to conventional therapy. Here, we show that CSCs express a type I receptor tyrosine kinase-like orphan receptor (ROR1), which is expressed during embryogenesis and by many different cancers, but not normal postpartum tissues. Ovarian cancers with high levels of ROR1 had stem cell-like gene-expression signatures.

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments.

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR.

Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21(WAF1/Cip1)-dependent mechanism independent of functional p53.

Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21).

MicroRNA-155 influences B-cell receptor signaling and associates with aggressive disease in chronic lymphocytic leukemia.

High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of ζ-chain-associated protein of 70 kD. CLL with high-level miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect.