Anna Shvartsur

Trop2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. It is an intracellular calcium signal transducer that is differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion, and survival. It has stem cell-like qualities. Trop2 is expressed in many normal tissues, though in contrast, it is overexpressed in many cancers and the overexpression of Trop2 is of prognostic significance. Several ligands have been proposed that interact with Trop2. Trop2 signals the cells via different pathways and it is transcriptionally regulated by a complex network of several transcription factors. Trop2 expression in cancer cells has been correlated with drug resistance. Several strategies target Trop2 on cancer cells that include antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. The in vitro studies and pre-clinical studies, using these various therapeutic treatments, have resulted in significant inhibition of tumor cell growth both in vitro and in vivo in mice. A clinical study is underway using IMMU-132 (hrS7 linked to SN38) in patients with epithelial cancers. This review describes briefly the various characteristics of cancer cells overexpressing Trop2 and the potential application of Trop2 as both a prognostic biomarker and as a therapeutic target to reverse resistance.

Multiple myeloma (MM) is a clonal plasma-cell neoplastic disorder arising from an indolent premalignant disease known as monoclonal gammopathy of undetermined significance (MGUS). MM is a biologically complex heterogeneous disease reflected by its variable clinical responses of patients receiving the same treatment. Therefore, a molecular identification of stage-specific biomarkers will support a more individualized precise diagnostic/prognostic approach, an effective therapeutic regime, and will assist in the identification of novel therapeutic molecular targets. The metastatic suppressor/anti-resistance factor Raf-1 kinase inhibitor protein (RKIP) is poorly expressed in the majority of cancers and is often almost absent in metastatic tumors. RKIP inhibits the Raf/MEK/ERK1/2 and the NF-κB pathways. Whereby all tumors examined exhibited low levels of RKIP, in contrast, our recent findings demonstrated that RKIP is overexpressed primarily in its inactive phosphorylated form in MM cell lines and patient-derived tumor tissues. The underlying mechanism of RKIP overexpression in MM, in contrast to other tumors, is not known. We examined transcriptomic datasets on Oncomine platform (Life Technologies) for the co-expression of RKIP and other gene products in both pre-MM and MM. The transcription of several gene products was found to be either commonly overexpressed (i.e., RKIP, Bcl-2, and DR5) or underexpressed (i.e., Bcl-6 and TNFR2) in both pre-MM and MM. Noteworthy, a significant inverse correlation of differentially expressed pro-apoptotic genes was observed in pre-MM: overexpression of Fas and TNF-α and underexpression of YY1 versus expression of anti-apoptotic genes in MM: overexpression of YY1 and underexpression of Fas and TNF-α. Based on the analysis on mRNA levels and reported studies on protein levels of the above various genes, we have constructed various schemes that illustrate the possible cross-talks between RKIP (active/inactive) and the identified gene products that underlie the mechanism of RKIP overexpression in MM. Clearly, such cross-talks would need to be experimentally validated in both MM cell lines and patient-derived tumor tissues. If validated, the differential molecular signatures between pre-MM and MM might lead to a more precise diagnosis/prognosis of the disease and disease stages and will also identify novel molecular therapeutic targets for pre-MM and MM.

Abstract Introduction: Macrophages polarize into pro-inflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. M2 cells promote tumor growth and metastasis through secretion of growth factors. However, crosstalk between tumor cells and macrophages in the development of M2 polarization has not been well demonstrated. We evaluated the proportion of M2 macrophages in bone marrow (BM) from patients (pts) with multiple myeloma (MM), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in the cell growth and differentiation of macrophages, we evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC) analysis, we determined the proportion of M1 and M2 macrophages in BM biopsies from MM pts with progressive disease and in remission. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells, following a standard IFC protocol. Monocyte/macrophage phenotypes for M1 and M2 subtypes in mononuclear cells isolated from MM BM aspirates were also examined using flow cytometric analysis with these same antibodies. Human monocytes isolated from normal subjects or the THP1 monocyte cell line were co-cultured with MM cell lines or primary MM tumor cells with or without exposure to low concentrations (IC20) of ruxolitinib (RUX) using Transwell plates. The percentage of M1 and M2 macrophages was determined using flow cytometric analysis. Total RNA was extracted from monocytes followed the manufacturer’s directions. Quantitation PCR were measured with TaqMan technology performed in an OneStepPlus instrument. Results: IFC demonstrated that the percentage of M2 macrophages (CD36+/ARG1+) was markedly increased in BM sections from MM pts with progressive disease compared with those pts in remission. The flow cytometric data also showed the percentage of M2 (CD36+/ ARG1+) macrophages in BM was significantly increased in MM patients with progressive disease (n=20) compared to those in remission (n=9; P=0.005) whereas there was no significant difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. The results of both RT-PCR and Quantitation PCR showed Trib1 gene expression levels were higher among patients with progressive disease compared to those in remission. In contrast, the gene expression of Trib2 and Trib3 was not related to the MM patient’s clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured MM cell lines or fresh MM tumor cells with purified healthy human monocytes using Transwell plates. The percentage of M2 cells markedly increased whereas the proportion of M1 cells decreased. The expression of Trib1 increased during the 7 days of co-culture whereas there was no change in the expression of Trib2 and Trib3. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages markedly increased after 7 days of incubation. It has been reported that Trib-1 mediated regulation of the MAPK/ERK pathway in a murine model and the Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT3 whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Thus, we investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. The percentage of M2 cells was decreased when the monocytes that were co-cultured with MM tumor cells were treated with a low concentration (IC20) of RUX. Trib1 gene expression of the monocytes treated with RUX was also reduced comparing to the cells untreated with JAK2 inhibitor. Conclusion: Our results show that MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. M2 cells are present at high levels in BM derived from MM pts with progressive disease compared to those in remission. Notably, the JAK2 inhibitor RUX shows inhibition of both M2 macrophage polarization and Trib1 gene expression in MM, and these results suggest this drug may be effective for treating MM. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Waldenström's macroglobulinemia (WM) is an incurable B-cell lymphoplasmacytic lymphoma. B-cell maturation antigen (BCMA) serves as one of the receptors for B-cell activating factor (BAFF) or a proliferation-inducing ligand (APRIL), which are members of the tumor necrosis factor (TNF) family that can induce activation of NF-κB and promote survival of B cells, including neoplastic B cells. We previously showed that serum BCMA levels are elevated in multiple myeloma (MM) patients, and correlated with disease status and overall survival (OS). In this study, we sought to determine whether BCMA levels are elevated in the serum of WM patients, track with conventional WM tumor markers, and correlate with disease status and OS. Methods: Data was obtained on a total of 67 WM patients who received treatment in one of two clinics that specialize in the treatment of WM. Serum BCMA levels were determined with a polyclonal anti-BCMA antibody using an ELISA (R&D Systems, Minneapolis, MN). Mann-Whitney analysis was used to measure differences in serum BCMA levels between WM patients and healthy subjects as well as the clinical status of WM patients. Using Kaplan-Meier analysis, OS (median follow up 5.1 years) in WM patients was measured from the time of the first assessment of serum BCMA levels in each patient. In addition, serum BCMA levels were compared to serum M-protein and IgM levels in 12 patients, serially, during their individual courses of treatment. Results: We compared serum BCMA levels for all 67 patients, including 27 untreated WM patients to healthy subjects (n = 76). Serum BCMA levels were markedly elevated in all WM patients (median 79.53 ng/mL), including those who were untreated (median 82.72 ng/mL) versus healthy subjects (median 6.32 ng/mL, p < 0.0001 for both comparisons). Serum BCMA values were compared to both serum M-protein and IgM levels in 12 WM patients during their disease course. Serum BCMA levels consistently correlated with changes in both of these established WM serum markers during their course of disease. Serum BCMA levels of WM patients also correlated with patient disease status at the time of sample collection. Specifically, serum from patients achieving > partial response (PR, n = 12) showed significantly lower levels of BCMA than samples from patients with either stable disease (SD, n = 8, p = 0.030) or progressive disease (PD, n = 21, p = 0.0004). OS (median 12.5 years) was determined among patients in the highest serum BCMA quartile (n=16; quartile range 128.91-812.48 ng/mL) and compared to levels in the lower three quartiles (n = 48; range 20.13-128.85 ng/mL). Notably, OS was shorter among patients in the top quartile compared to those in the other three quartiles (p = 0.02). Conclusions: This is the first study to demonstrate that serum BCMA levels are elevated in Waldenström's macroglobulinemia patients. Importantly, serum BCMA levels correlated with both serum IgM and M-protein levels, as well as tracked with these established WM biomarkers for individual patients during their course of disease. Additionally, patients with levels of BCMA in the highest quartile exhibited shorter OS. Thus, serum BCMA represents a new serum biomarker to predict outcome and monitor patients with Waldenström's macroglobulinemia. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV). Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs. Results: In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone. Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. Disclosures No relevant conflicts of interest to declare.