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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 − / − γ c − / − 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 − / − γ c − / − 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.

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.

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 ROR1 P808A 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.

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. Furthermore, patients with ovarian cancers with high levels of ROR1 had higher rates of relapse and a shorter median survival than patients with ovarian cancers that expressed low-to-negligible amounts of ROR1 . We found that ROR1-positive (ROR1 ⁺) cells isolated from primary tumor-derived xenografts (PDXs) also expressed aldehyde dehydrogenase 1 (ALDH1) and had a greater capacity to form spheroids and to engraft immune-deficient mice than did ROR1-negative (ROR1 ᴺᵉᵍ) ovarian cancer cells isolated from the same tumor population. Treatment with UC-961, an anti-ROR1 mAb, or shRNA silencing of ROR1 inhibited expression of the polycomb ring-finger oncogene, Bmi-1, and other genes associated with the epithelial–mesenchymal transition. Moreover, shRNA silencing of ROR1, depletion of ROR1 ⁺ cells, or treatment with UC-961 impaired the capacity of ovarian cancer cells to form spheroids or tumor xenografts. More importantly, treatment with anti-ROR1 affected the capacity of the xenograft to reseed a virgin mouse, indicating that targeting ROR1 may affect CSC self-renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which contributes to their capacity to form tumors, making ROR1 a potential target for the therapy of patients with ovarian cancer. Significance This study demonstrates that the oncoembryonic surface antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1), is expressed on human ovarian cancer stem cells (CSCs), on which it seems to play a functional role in promoting migration/invasion or spheroid formation in vitro and tumor engraftment in immune-deficient mice. Treatment with a humanized mAb specific for ROR1 (UC-961) could inhibit the capacity of ovarian cancer cells to migrate, form spheroids, or engraft immune-deficient mice. Moreover, such treatment inhibited the growth of tumor xenografts, which in turn had a reduced capacity to engraft immune-deficient mice and were relatively depleted of cells with features of CSC, suggesting that treatment with UC-961 could impair CSC renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which may be targeted for anti-CSC therapy.

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/m 2 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.

Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a \"liquid biopsy\" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Significance IGHV mutation status is a well established prognostic factor in chronic lymphocytic leukemia, and also provides crucial insights into tumor cell biology and function. Currently, determination of IGHV transcript sequence, from which mutation status is calculated, requires a specialized laboratory procedure. RNA sequencing is a method that provides high resolution, high dynamic range transcriptome data that can be used for differential expression, isoform discovery, and variant determination. In this paper, we demonstrate that unselected next-generation RNA sequencing can accurately determine the IGH@ sequence, including the complete sequence of the complementarity-determining region 3 (CDR3), and mutation status of CLL cells, potentially replacing the current method which is a specialized, single-purpose Sanger-sequencing based test. 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. Cancers arising from mature B cells are characterized by clonal production of Ig heavy ( IGH@ ) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@ V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was out-of-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.

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 (ROR1 Hi ). ROR1 Hi 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.

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.