LPS-CXCL10 Predicts Responses to Bortezomib in Myeloma Patients

To identify predictive biomarkers for clinical responses to bortezomib treatment, 0.06 mL of each whole blood without any cell separation procedures was stimulated ex vivo using five agents, and eight mRNAs were quantified. In six centers, heparinized peripheral blood was prospectively obtained from 80 previously treated or untreated, symptomatic multiple myeloma (MM) patients with measurable levels of M-proteins. The blood sample was procured prior to treatment as well as 2-3 days and 1-3 weeks after the first dose of bortezomib, which was intravenously administered biweekly or weekly, during the first cycle. Six stimulant-mRNA combinations; that is, lipopolysaccharide (LPS)-granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS-CXCL chemokine 10 (CXCL10), LPS-CCL chemokine 4 (CCL4), phytohemagglutinin-CCL4, zymosan A (ZA)-GMCSF and ZA-CCL4 showed significantly higher induction in the complete and very good partial response group than in the stable and progressive disease group, as determined by both parametric (t-test) and non-parametric (unpaired Mann-Whitney test) tests. Moreover, LPS-induced CXCL10 mRNA expression was significantly suppressed 2-3 days after the first dose of bortezomib in all patients, as determined by both parametric (t-test) and non-parametric (paired Wilcoxon test) tests, whereas the complete and very good partial response group showed sustained suppression 1-3 weeks after the first dose. Thus, pretreatment LPS-CXCL10 mRNA and/or the six combinations may serve as potential biomarkers for the response to bortezomib treatment in MM patients

Lipopolysaccharide-Induced CXCL10 mRNA Level and Six Stimulant-mRNA Combinations in Whole Blood: Novel Biomarkers for Bortezomib Responses Obtained from a Prospective Multicenter Trial for Patients with Multiple Myeloma.

To identify predictive biomarkers for clinical responses to bortezomib treatment, 0.06 mL of each whole blood without any cell separation procedures was stimulated ex vivo using five agents, and eight mRNAs were quantified. In six centers, heparinized peripheral blood was prospectively obtained from 80 previously treated or untreated, symptomatic multiple myeloma (MM) patients with measurable levels of M-proteins. The blood sample was procured prior to treatment as well as 2-3 days and 1-3 weeks after the first dose of bortezomib, which was intravenously administered biweekly or weekly, during the first cycle. Six stimulant-mRNA combinations; that is, lipopolysaccharide (LPS)-granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS-CXCL chemokine 10 (CXCL10), LPS-CCL chemokine 4 (CCL4), phytohemagglutinin-CCL4, zymosan A (ZA)-GMCSF and ZA-CCL4 showed significantly higher induction in the complete and very good partial response group than in the stable and progressive disease group, as determined by both parametric (t-test) and non-parametric (unpaired Mann-Whitney test) tests. Moreover, LPS-induced CXCL10 mRNA expression was significantly suppressed 2-3 days after the first dose of bortezomib in all patients, as determined by both parametric (t-test) and non-parametric (paired Wilcoxon test) tests, whereas the complete and very good partial response group showed sustained suppression 1-3 weeks after the first dose. Thus, pretreatment LPS-CXCL10 mRNA and/or the six combinations may serve as potential biomarkers for the response to bortezomib treatment in MM patients

Bortezomib-induced inhibition of LPS-induced <i>CXCL10</i> mRNA <i>ex vivo</i>.

<p>Peripheral blood obtained from 3 healthy volunteers was pre-treated with various concentrations of bortezomib for 1 h and then further stimulated with LPS or PBS (as the control) for an additional 4 h. The fold increase in (A) <i>ACTB</i> and (B) <i>CXCL10</i> expression is shown. Each symbol represents a single individual.</p

<i>Ex vivo</i> mRNA induction in blood obtained prior to bortezomib treatment.

<p>The fold increase in (A) LPS-induced <i>GMCSF</i>, (B) ZA-induced <i>GMCSF</i>, (C) LPS-induced <i>CXCL10</i> (top panel), (D) PHA-induced <i>CCL4</i>, (E) LPS-induced <i>CCL4</i> and (F) ZA-induced <i>CCL4</i> (lower panel) mRNA in the CR, VGPR, PR, SD and PD groups is shown. The statistically significant difference between the CR+VGPR and SD+PD groups is shown. t: Student’s <i>t</i>-test, M: Mann-Whitney test. Dotted line: fold increase = 3. Samples showing a fold increase in <i>ACTB</i> (which was > 3) were removed from the analysis. Horizontal bars: the mean values.</p

Patients Demographic and Baseline Characteristics.

<p>CR, complete response; ISS, International Staging System; NE, not evaluable; PD, progressive disease; SD, stable disease; VGPR, very good partial response.</p><p>*Excluded were three patients not evaluable for response.</p><p>^†According to the International Uniform Response Criteria (Durie et al, 2006).</p><p>^‡One patient died of progressive disease early, another received additional chemotherapy, and the third committed suicide.</p><p>Patients Demographic and Baseline Characteristics.</p

Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM

LPS-induced <i>CXCL10</i> expression before and after bortezomib treatment.

<p>Each point/line represents the fold increase in LPS-induced <i>CXCL10</i> expression in each patient in the (A) CR+VGPR, (B) PR and (C) SD+PD groups. The statistically significant difference between the pretreatment (D0) and 2–3 days (D2-3) or 1–3 weeks (W1-3) after intravenous administration of the first dose of bortezomib during the first cycle groups is shown. t: Student’s <i>t</i>-test, W: Wilcoxon test.</p

TAS-116 effects on cell viability and RAS-RAF-MEK-ERK signaling in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with TAS-116 (0–5 μM) for 24, 48, or 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with the indicated doses of TAS-116 for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form.</p

Combination of TAS-116 plus tipifarnib, dabrafenib, or AZD6244 blocks the growth stimulatory effect of the bone marrow microenvironment.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with TAS-116 (1 μM) either alone or in combination with tipifarnib (NCI-H929: 2 μM, INA6: 0.5 μM, MM.1S: 2 μM, RPMI-8226: 2 μM), dabrafenib (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 10 μM, RPMI-8226: 10 μM), or AZD6244 (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 20 μM, RPMI-8226: 20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; Tipi, tipifarnib; Dabra, dabrafenib; AZD, AZD6244. (B) MM.1S and NCI-H929 cells were cultured with TAS-116 (2 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244 for 24 h in the presence or absence of BMSC supernatant. Whole-cell lysates were subjected to western blotting using PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form; SN, supernatant. (C) MM.1S cells were cultured with TAS-116 (1 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244; and NCI-H929 cells were cultured with TAS-116 (1 μM), AZD6244 (10 μM), or TAS-116 plus AZD6244 for 48 h in the presence or absence of BMSC supernatant. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; AZD, AZD6244; SN, supernatant (*: <i>P</i> < 0.05; **: <i>P</i> < 0.01).</p