Jessie-F. Fecteau

Targeting ROR1 Inhibits Epithelial-Mesenchymal Transition and Metastasis

Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across the anatomic barriers and colonize distant organs. Here, we show an association between metastasis and expression of a type I receptor tyrosine kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal postpartum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high levels of ROR1 were more likely to have gene expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast cancer cell lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g., vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g., CK-19), and tight junction proteins (e.g., ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immunodeficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced the expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a monoclonal antibody specific for ROR1 induced downmodulation of vimentin and inhibited cancer cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis and that antibodies targeting ROR1 can inhibit cancer progression and metastasis.

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). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.

Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44

Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid. We found that a humanized mAb specific for CD44 (RG7356) was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, RG7356 could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspase-dependent apoptosis, independent of complement or cytotoxic effector cells. The cytotoxic effect of this mAb was not mitigated when the CLL cells were cocultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-µ. RG7356 induced rapid internalization of CD44 on CLL cells at 37 °C, resulting in reduced expression of ZAP-70, which we found was complexed with CD44. Administration of this mAb at a concentration of 1 mg/kg to immune-deficient mice engrafted with human CLL cells resulted in complete clearance of engrafted leukemia cells. These studies indicate that this mAb might have therapeutic activity, particularly in patients with CLL that express ZAP-70.

Sorafenib-Induced Apoptosis of Chronic Lymphocytic Leukemia Cells Is Associated with Downregulation of RAF and Myeloid Cell Leukemia Sequence 1 (Mcl-1)

We have previously shown that sorafenib, a multikinase inhibitor, exhibits cytotoxic effects on chronic lymphocytic leukemia (CLL) cells. Because the cellular microenvironment can protect CLL cells from drug-induced apoptosis, it is important to evaluate the effect of novel drugs in this context. Here we characterized the in vitro cytotoxic effects of sorafenib on CLL cells and the underlying mechanism in the presence of marrow stromal cells (MSCs) and nurselike cells (NLCs). One single dose of 10 µmol/L or the repeated addition of 1 µmol/L sorafenib caused caspase-dependent apoptosis and reduced levels of phosphorylated B-RAF, C-RAF, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia sequence 1 (Mcl-1) in CLL cells in the presence of the microenvironment. We show that the RAF/mitogen-activated protein kinase kinase (MEK)/ERK pathway can modulate Mcl-1 expression and contribute to CLL cell viability, thereby associating sorafenib cytotoxicity to its impact on RAF and Mcl-1. To evaluate if the other targets of sorafenib can affect CLL cell viability and contribute to sorafenib-mediated cytotoxicity, we tested the sensitivity of CLL cells to several kinase inhibitors specific for these targets. Our data show that RAF and vascular endothelial growth factor receptor (VEGFR) but not KIT, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) are critical for CLL cell viability. Taken together, our data suggest that sorafenib exerts its cytotoxic effect likely via inhibition of the VEGFR and RAF/MEK/ERK pathways, both of which can modulate Mcl-1 expression in CLL cells. Furthermore, sorafenib induced apoptosis of CLL cells from fludarabine refractory patients in the presence of NLCs or MSCs. Our results warrant further clinical exploration of sorafenib in CLL.

Structure and function of the hematopoietic cancer niche: focus on chronic lymphocytic leukemia

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy characterized by the accumulation of mature monoclonal CD5-positive B cells in the blood, secondary lymphoid tissues, and marrow. The infiltration of CLL cells in lymphoid tissues is a key element of disease pathogenesis. It is in such tissues that are found the microenvironments that provide CLL cells protection from spontaneous and/or drug-induced apoptosis. CLL cells actively shape their microenvironment by producing cytokines and chemokines, and by subverting normal accessory cells to promote leukemia-cell survival, proliferation, and escape from immune detection. In this review, we discuss how CLL cells disrupt the niches required for normal hematopoiesis or immune function and subvert normal cells in the microenvironment to support neoplastic cell growth and survival.

Impact of oxygen concentration on growth of mesenchymal stromal cells from the marrow of patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells interact in the marrow with mesenchymal stromal cells (MSCs), which can enhance CLL-cells’ resistance to spontaneous or drug-induced apoptosis. Here we examined the effect of oxygen on the growth and function of MSCs from marrow aspirates of CLL patients. Cultures in ambient oxygen provided for poor recovery and growth of MSCs, which developed features of cell senescence. However, MSCs were propagated readily from the same cells when they were cultured at a physiologic oxygen concentration of 5%. Such MSCs promoted short-term CLL-cell survival in either 5% or ambient O2. However, longer-term CLL-cell survival was enhanced when the cocultures were maintained in 5% O2 versus 21% O2 because of increased MSC proliferation and production of soluble prosurvival factors, such as CXCL12. This study establishes the importance of physiologic oxygen concentration in the propagation and function of MSCs derived from marrow aspirates of CLL patients in vitro.

Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia.

Mutations in genes encoding the spliceosome machinery recently have been described in hematological malignancies,1, 2 particularly in myelodysplastic syndrome (MDS) and chronic lymphocytic leukemia (CLL). These mutations can occur in genes encoding different components of the spliceosome.3 However, the most frequent of such mutations observed in CLL occurs in the gene-encoding splicing factor 3 subunit 1 (SF3B1), a core component of the RNA splicing machinery,1, 4, 5, 6 and is found in 5–17% of CLL patients.5, 6, 7 SF3B1 mutations have been associated with a relatively poor prognosis in CLL,6, 7, 8 but appear associated with a relatively good prognosis in MDS.2, 9, 10, 11 Although initially proposed to represent a driver mutation in CLL,4 a recent study noted two cases in which the CLL cells appeared to have acquired SF3B1 mutations during clonal evolution.7 In this report, we investigated the progression of SF3B1 mutations in the CLL B cells over time in an attempt to elucidate whether there exists subclonal evolution involving SF3B1 mutations in CLL. Accumulation of CLL cells harboring mutations in SF3B1 suggests that such subclones have some competitive advantage, which might account for accelerated progression of the disease in some patients over time. Alternatively, subclones of CLL cells might be selected during therapy, similar to what has been observed in mutations involving TP53 in CLL cells of patients treated with standard chemotherapy. To address these questions, we examined for SF3B1 mutations in the CLL cells of 545 patients. The CLL samples of 448 patients were acquired before any therapy, and 97 following treatment for CLL. We investigated for changes in the proportionate representation of subclones harboring SF3B1 mutations over time, with or without therapeutic intervention. Thirty-six cases (6.6%) had CLL cells with detectable mutations in the 545 cases examined. In all, 20 (56%) of these 36 cases had K700E and 6 had K666E/Q/N/T (6/36, 17%). As noted in another study,7 the proportion of cases that had SF3B1 mutations was significantly higher in the samples of patients who had received prior therapy (12.4%, 12/97) than in samples from patients with no prior therapy (5.4%, 24/448, P=0.02) (Supplementary Figure 1A). Cases found to have SF3B1 mutations more commonly were found to have unfavorable prognostic features than did cases without SF3B1 mutations, such as expression of unmutated immunoglobulin heavy chain variable region genes (75%, 27/36, P=0.0002), as reported previously.5 Furthermore, a higher proportion of cases with SF3B1 mutations also expressed ZAP-70 (75%, 27/36, P<0.0001) or CD38 (58%, 21/36, P=0.0007) than did cases without SF3B1 mutations (Supplementary Figure 1B). Patients with CLL cells harboring SF3B1 mutations were found to have a significantly shorter median treatment-free survival (3.0 versus 6.0 years, P<0.0001) and overall survival (OS, 11.4 versus 21.0 years, P=0.0021) than did patients with CLL cells lacking detectable SF3B1 mutations (Supplementary Figures 1C and D). Fluorescence in situ hybridization (FISH) data were available for 362 (71%) out of 509 CLL patients with leukemic cells without SF3B1 mutations and 33 (92%) out of 36 CLL patients with leukemic cells with SF3B1 mutations. A higher proportion of the SF3B1-mutated cases lacked detectable chromosomal abnormalities than did those cases without SF3B1 mutations (15/33, 45% versus 88/362, 24%, P=0.012). Regarding the genetic abnormalities detected by FISH (del17p, del11q, +12 and del13q), we did not observe any one genetic abnormality associated with cases that did or did not have SF3B1 mutations (Supplementary Figures 2A and B). We then examined serial samples from the patients identified as having SF3B1 mutations. Of 36 cases identified to have SF3B1 mutations, 20 cases were included in these analyses (Table 1) with time intervals between sample collections ranging from 6 to 61.5 months (median 23.7 months). We measured the height of the peak corresponding to the wild-type or mutant SF3B1 and calculated the proportionate representation of the height for the mutant allele of samples collected at different times (Figure 1). For some of the samples, this value exceeded 50% (for example, cases 2,12, 14 and 19). None of these samples had the most common SF3B1 mutation (for example, K700E), making it unlikely that these samples had the same mutation on both alleles. Rather it is more likely that each of these samples had cells harboring a mutation in SF3B1 and a deletion of the wild-type allele. Prior studies had identified uncommon cases of CLL that had deletions in 2q (the chromosomal location for SF3B1), which were not detected by conventional cytogenetics.12 For seven cases (no. 1–7), we noted significant increases in the proportionate representation of the SF3B1-mutant allele over time (Table 1). For three out of the seven cases (cases 2, 3 and 4), all serial samples were collected before therapy; for the other four cases, all serial samples were collected after therapy (Supplementary Table 1). These data indicate that the proportion of subclones harboring SF3B1 mutations can increase spontaneously over time independent of any therapeutic intervention. Figure 1 Evolution of SF3B1 mutations over time. (a–d) Representation of the serial sample sequence chromatograms for patient no. 1, 2, 3 and 4, respectively. The time intervals between each time point are indicated as well as the overall time span studied. ... Table 1 Twenty cases with SF3B1 mutations examined serially We studied p53 function in two out of these four cases (no. 1 and 6, Supplementary Figure 3), for which CLL cells were available at the second sample collection and after treatment. To evaluate p53 function, we monitored for expression of p53 and p21 after γ-irradiation via flow cytometry. We found γ-irradiation-induced p53 and p21 in each of these two samples, indicating functional TP53. As such, subclonal expansion of cells harboring mutations in SF3B1 can occur in cells that have not lost p53 function. For 19 cases out of the 20 cases analyzed serially, we conducted FISH analyses on the serial samples. We did not observe any changes between the serial samples for 15 (79%) cases, including 4 that had the largest increases in the proportionate representation of the SF3B1 allele over time in association with concurrent disease progression (Supplementary Table 1). However, in four cases, we observed one or two new cytogenetic abnormalities in the second sample (for example, for case 11, there was acquisition of del13q, for cases 17 and 18, there was acquisition of del17p and for case 14, there was acquisition of del13q and del17p). Patient no. 1 was treated with fludarabine, cyclophosphamide and rituximab in July 2004. The patient relapsed after achieving a partial response, developing progressive disease. Between sample collections, the patients absolute lymphocyte count (ALC) increased from 13 to 102 × 109/l, which was associated with an increase in the proportionate representation of the SF3B1 allele from 30 to 44%. Similarly, between sample collections of patient no.2, the ALC increased from 67 to 250 × 109/l, which was associated with an increase in the proportionate representation of the SF3B1 allele from 37 to 60%. Patient no. 3 experienced increases in ALC from 87 to 197 × 109/l between sample collections, for which we detected an increase in the proportionate representation of the SF3B1 allele from 16 to 43%. Finally, the ALC increased in patient no. 4 from 8 to 47 × 109/l between sample collections, for which we noted an increase in the proportionate representation of the SF3B1 allele from 24 to 39%. These data imply that subclones of CLL cells harboring mutant SF3B1 had either a higher growth rate and/or lower death rate than subclones lacking mutations in SF3B1, this potentially contributing to disease progression. For four cases with SF3B1 mutations (cases 6, 13, 16 and 17), we examined samples before and after therapy, which resulted in >50% reduction in ALC. For each of these cases, however, we did not observe a significant change in the proportionate representation of the mutant SF3B1 allele, indicating that such treatments did not have selective activity or inactivity for subclones with SF3B1 mutations. Taken together, this study reveals subclonal evolution involving cells with SF3B1 mutations in CLL. Our data indicate that the proportionate representation of cells harboring SF3B1 mutations can increase independent of therapy or loss of functional p53. Finally, the data presented here suggest that subclones with SF3B1 mutations do not necessarily have a selective advantage or disadvantage in the setting of effective cytoreductive therapy. Nevertheless, the prevalence of SF3B1 mutations appears higher among patients who already have undergone therapy. This might reflect the fact that treated patients more commonly have had longer disease histories, potentially providing greater time for emergence and subclonal evolution of CLL cells harboring SF3B1 mutations. Further studies with additional cases will be required to address the therapeutic implications of the SF3B1 mutations and subclonal evolution in this disease.

Abstract 975: MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling

MicroRNAs (miRNAs) can influence the expression levels of genes that can affect the clinical outcome of patients with chronic lymphocytic leukemia (CLL). In a well-defined cohort of 86 patients, we observed heterogeneity in the relative leukemia-cell expression of miR-155. Cases that expressed high-levels of miR-155 more frequently expressed zeta-associated protein of 70 kD (ZAP-70), used unmutated Ig heavy-chain-variable-region genes (IGHV), and had shorter treatment-free survival (TFS) and overall survival (OS) than cases with low-level expression of miR-155. Recursive partitioning allowed us to define a threshold for “high-level” expression of miR-155 (in copy numbers per CLL cell) that best associates with adverse clinical outcome. We examined the potential basis for this association. One of the genes targeted by miR-155 encodes the phosphatidylinositol polyphosphate 5-phosphatase SHIP-1, which can dephosphorylate proteins that had been activated in response to B-cell receptor (BCR) ligation. We found that CLL cells with “high-level” miR-155 expressed significantly lower levels of SHIP-1 protein and were more sensitive to ligation of surface-µ than CLL cells with low-levels of miR-155. Transfecting CLL cells with miR-155 reduced their expression of SHIP-1 and enhanced the stimulatory-response to surface-µ ligation. Conversely, transfection of CLL cells with a miR-155 inhibitor had the opposite effects. The enhanced sensitivity to BCR-ligation of cells expressing “high-level” miR-155 may account for its association with adverse clinical outcome in patients with CLL. However, within any one patient we also find heterogeneity in the expression levels of miR-155. By GEO data analysis (GSE21029) we found that CLL cells in blood expressed lower levels of miR-155 than did CLL cells in lymphoid tissues, where they interact with supportive cells, such as Nurselike cells or activated T cells, which can provide survival- or growth-promoting signals via expression of BAFF/APRIL or CD40L, respectively. We found that stimulation of CLL cells in vitro via CD40 ligation or exogenous BAFF could induce higher expression of miR-155, leading to reduced leukemia-cell expression of SHIP-1 and enhanced sensitivity to surface-µ ligation. Moreover, we found that the “proliferative” subgroup of blood CD5brightCXCR4dim CLL cells, which represent CLL cells newly released from the tissue microenvironment, expressed higher level of miR-155 and lower levels of SHIP-1 protein, and were more sensitive to surface-µ ligation than the “resting” subgroup of blood CD5dimCXCR4bright CLL cells, which represent cells that may be due to enter the tissue microenvironment. As such, this study also demonstrates that miR-155 could be upregulated by signals in the CLL microenvironment, leading to lower-level expression of SHIP-1, and enhanced stimulation in response to BCR ligation. Citation Format: Liguang Chen, Bing Cui, Suping Zhang, Marek Mraz, Jessie-F. Fecteau, Jian Yu, Ling Zhang, Lei Bao, Laura Rassenti, Karen Messer, Carlo Croce, Thomas Kipps. MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 975. doi:10.1158/1538-7445.AM2014-975