Emanuela M. Ghia

Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy

The Brutons tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

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.

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. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5(bright)CXCR4(dim) cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5(dim)CXCR4(bright) cells of the same patient. Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL.

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

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. 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 (ROR1Neg) 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.

Use of IGHV3-21 in chronic lymphocytic leukemia is associated with high-risk disease and reflects antigen-driven, post-germinal center leukemogenic selection.

We examined the chronic lymphocytic leukemia (CLL) cells of 2457 patients evaluated by the CLL Research Consortium (CRC) and found that 63 (2.6%) expressed immunoglobulin (Ig) encoded by the Ig heavy-chain-variable-region gene (IGHV), IGHV3-21. We identified the amino acid sequence DANGMDV (motif-1) or DPSFYSSSWTLFDY (motif-2) in the Ig heavy-chain (IgH) third complementarity-determining region (HCDR3) of IgH, respectively, used by 25 or 3 cases. The IgH with HCDR3 motif-1 or motif-2, respectively, was paired with Ig light chains (IgL) encoded by IGLV3-21 or IGKV3-20, suggesting that these Ig had been selected for binding to conventional antigen(s). Cases that had HCDR3 motif-1 had a median time from diagnosis to initial therapy comparable with that of cases without a defined HCDR3 motif, as did cases that used mutated IGHV3-21 (n = 27) versus unmutated IGHV3-21 (n = 30). Of 7 examined cases that used Ig encoded by IGHV3-21/IGLV3-21, we found that 5 had a functionally rearranged IGKV allele that apparently had incurred antigendriven somatic mutations and subsequent rearrangement with KDE. This study reveals that CLL cells expressing IGHV3-21/IGLV3-21 most likely were derived from B cells that had experienced somatic mutation and germinal-center maturation in an apparent antigen-driven immune response before undergoing Ig-receptor editing and after germinal-center leukemogenic selection.

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.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen.

Evidence of biased immunoglobulin variable gene usage in highly stable B-cell chronic lymphocytic leukemia

Recognition of biased immunoglobulin variable (IgV) gene usage in B-cell chronic lymphocytic leukemia (B-CLL) may yield insight into leukemogenesis and may help to refine prognostic categories. We explored Ig variable heavy (VH) and light (VL) chain gene usage in highly stable and indolent B-CLL (n=25) who never required treatment over 10 or more years. We observed an unexpectedly high usage of mutated VH3-72 (6/25; 24.0%), a gene that was otherwise rare in B-CLL (7/805; 0.87%; P<0.01), including mutated cases (6/432; 1.39%; P<0.01) and was exceptional among indolent (1/230, 0.435%; P<0.01), and aggressive B-cell lymphomas (0/105; P<0.01). Three of six VH3-72 B-CLL cases utilized the same VL Vκ4-1 gene. Two VH3-72 B-CLL cases had highly homologous VH complementarity determining regions 3 (CDR3s), encoding Cys-XXXX-Cys domains, and utilized Vκ4-1 genes with homologous IgVL CDR3s. An identical threonine to isoleucine change at codon 84 of VH3-72 framework region 3 (FR3) recurred in four cases of highly stable VH3-72 B-CLL. This mutation is expected to cause a conformational change of FR3 proximal to CDR3 that might critically affect high-affinity antigen binding. B-cell receptors encoded by VH3-72 may identify a specific B-CLL group and be implicated in leukemogenesis through an antigen-driven expansion of B cells.

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.

ROR1 can interact with TCL1 and enhance leukemogenesis in Eμ-TCL1 transgenic mice.

Significance Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type 1 protein expressed on chronic lymphocytic leukemia (CLL) B cells, but not on normal postpartum tissues. This study demonstrates that ROR1 can contribute to leukemogenesis and can bind to T-cell leukemia 1 (TCL1), a known coactivator of AKT. ROR1 can accelerate leukemogenesis when expressed together TCL1, leading to increased activation of AKT and enhanced leukemia-cell proliferation and resistance to apoptosis. Treatment of ROR1-expressing leukemia cells with an anti-ROR1 mAb could down-modulate ROR1, reduce phospho-AKT, and impair their capacity to engraft syngeneic mice. Collectively, our data demonstrate that ROR1 accelerates development/progression of leukemia and may be targeted for therapy of patients with CLL. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen found on chronic lymphocytic leukemia (CLL) B cells, but not on normal adult tissues. We generated transgenic (Tg) mice with human ROR1 regulated by the murine Ig promoter/enhancer. In contrast to nontransgenic littermates, such animals had B-cell–restricted expression of ROR1 and could develop clonal expansions of ROR1brightCD5+B220low B cells resembling human CLL at ≥15 mo of age. Because immune-precipitation and mass spectrometry studies revealed that ROR1 could complex with T-cell leukemia 1 (TCL1) in CLL, we crossed these animals with Eµ-TCL1-Tg (TCL1) mice. Progeny with both transgenes (ROR1 × TCL1) developed CD5+B220low B-cell lymphocytosis and leukemia at a significantly younger median age than did littermates with either transgene alone. ROR1 × TCL1 leukemia B cells had higher levels of phospho-AKT than TCL1 leukemia cells and expressed high levels of human ROR1, which we also found complexed with TCL1. Transcriptome analyses revealed that ROR1 × TCL1 leukemia cells had higher expression of subnetworks implicated in embryonic and tumor-cell proliferation, but lower expression of subnetworks involved in cell–cell adhesion or cell death than did TCL1 leukemia cells. ROR1 × TCL1 leukemia cells also had higher proportions of Ki-67–positive cells, lower proportions of cells undergoing spontaneous apoptosis, and produced more aggressive disease upon adoptive transfer than TCL1 leukemia cells. However, treatment with an anti-ROR1 mAb resulted in ROR1 down-modulation, reduced phospho-AKT, and impaired engraftment of ROR1 × TCL1 leukemia cells. Our data demonstrate that ROR1 accelerates development/progression of leukemia and may be targeted for therapy of patients with CLL.