Journal: Nature Communications

Article Title: The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

doi: 10.1038/s41467-024-51442-2

Figure Lengend Snippet: a Representative western blots of MM.1S and MM.1S-pomalidomide-resistant cells cultured with or without TGFβ1 (10 ng/mL) treatment. Whole cell lysates were prepared and probed with the antibodies indicated in Fig. , along with Bcl-2 and cleaved caspase. Densitometric values for cereblon, IKZF1 and IKZF3 are relative to the value determined in WT (parental) cells in the absence of TGFβ. Densitometric values for total SMAD2/3 and total phospho-ERK1/2 are relative that the values measured in the absence of TGFβ in WT (parental) cells. Densitometric values for phospho-SMAD2/3 were determined relative to the total SMAD2/3 value determined with the same cells (either WT or pomalidomide-resistant) with or without TGFβ. Similarly, densitometric values for phospho-ERK1/2 were determined relative to the total ERK1/2 value determined with the same cells (WT or pomalidomide-resistant) with or without TGFβ. Experiments were independently performed twice. b Shown is the dose-dependent effect of pomalidomide and vactosertib on MM1.S parental and pomalidomide-resistant cells. Cells were treated with drugs at each indicated concentrations in triplicate and viability then determined using the XTT assay. Values shown represent the arithmetic mean of three independent experiments. c Concentration dependent combined effect of vactosertib and pomalidomide on MM.1S parental, pomalidomide-resistant and patient CD138 + cells. Cells were treated with drugs at each indicated concentrations in triplicate and viability then determined using the XTT assay. Values shown represent the arithmetic mean of three independent experiments. Green boxes indicate highest viability, yellow boxes intermediate viability and red boxes lowest viability. Source data are provided.

Article Snippet: CD138 + cells were isolated from the BMMC fraction using the EasySep TM human CD138 positive selection kit II (StemCell Technology, Cambridge, MA).

Techniques: Western Blot, Cell Culture, XTT Assay, Concentration Assay

Journal: Nature Communications

Article Title: The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

doi: 10.1038/s41467-024-51442-2

Figure Lengend Snippet: a Western blot analysis of MM1.S parental and pomalidomide-resistant cells treated with vactosertib and pomalidomide alone or in combination. Cells were treated with drugs as indicated, lysates prepared and probed with antibodies to phospho-ERK1/2, total ERK1/2, Bcl-2 and full-length (FL), cleaved caspase-3 and GADPH. Experiments were independently performed twice. b Combined effect of vactosertib and pomalidomide on MM1.S and MM1.S- pomalidomide-resistant cells. Cells (1 × 10 6 /assay) were incubated with drugs for 20 h, washed with PBS, lysates prepared, probed using antibodies specific to annexin-V (BD Biosciences, 556419) and propidium iodide (PI) (Invitrogen, P21493) and analyzed by flow cytometry (Attune NxT). Relative % of annexin-V+/PI+ cells represents the percent of drug treated MM cells relative to untreated cells. c Combined effect of vactosertib and pomalidomide on patient BM CD138 + cells. Cells (~1 × 10 5 /assay) were incubated with drugs for 20 h, washed with PBS, probed using antibodies specific to annexin-V (BD Biosciences, 556419) and PI and analyzed by flow cytometry. Annexin-V+/PI+ cells represent the percent of CD138 + cells positive for both annexin-V and PI relative to untreated cells. In ( b ) and ( c ), data are representative of three independent experiments. Error bars represent the SD of the mean (SEM). Two-way ANOVA was conducted to investigate potential interactions between more than two variables in ( b ) and ( c ).

Article Snippet: CD138 + cells were isolated from the BMMC fraction using the EasySep TM human CD138 positive selection kit II (StemCell Technology, Cambridge, MA).

Techniques: Western Blot, Incubation, Flow Cytometry

Journal: Nature Communications

Article Title: The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

doi: 10.1038/s41467-024-51442-2

Figure Lengend Snippet: a Shown is the relative MFI value for the expression of each immunosuppressive marker (PD-1, TIM-3, BTLA, and LAG-3) on CD8 + T-cells isolated from healthy peripheral blood. T-cells were cultured in the presence of TGFβ1 at indicated concentrations. CTLA-4 was not detected by immunostaining of healthy human T-cells. Error bars represent the SD of the mean. b Shown are relative MFI values after staining for PD-1, TIM-3 and BTLA on MM patient CD8 + T-cells. Error bars represent the SD of the mean. c Effect of vactosertib on PD-L1 and PD-L2 expression on MMCLs. MMCLs were treated with vactosertib and pomalidomide as indicated for 72 h. Cells were then stained with PD-L1 and PD-L2 antibodies for 20 min and analyzed by flow cytometry. d Effect of vactosertib on PD-L1 and PD-L2 expression on MM patient BM-derived CD138 + cells. Patient CD138 + cells were treated with vactosertib and/or pomalidomide at various concentrations for 48 h. Cells were then stained with PD-L1 and PD-L2 specific antibodies simultaneously for 20 min and analyzed by flow cytometry. Statistical comparisons were made by comparing drug treated to untreated cells. In ( a – d ), values represent the average of triplicate measurements. Two-way ANOVA was conducted to investigate potential interactions between more than two variables in ( a – d ). Source data are provided as a Source Data file.

Article Snippet: CD138 + cells were isolated from the BMMC fraction using the EasySep TM human CD138 positive selection kit II (StemCell Technology, Cambridge, MA).

Techniques: Expressing, Marker, Isolation, Cell Culture, Immunostaining, Staining, Flow Cytometry, Derivative Assay

Journal: Nature Communications

Article Title: The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

doi: 10.1038/s41467-024-51442-2

Figure Lengend Snippet: a Effect of TGFβ1 on the viability of patient CD8 + T-cells. Cells (5000/well) were treated as indicated for 24 h. Activated-XTT assay solution was added and plates were incubated in the dark for 3 h. Relative cell viability was quantified by measuring the absorbance at 450 nm on a SpectraMax i3x multi-mode microplate reader. b Effect of vactosertib on the viability of patient CD8 + T-cells in the presence of TGFβ1. Cells (5000/well) were treated as indicated for 24 h. Activated-XTT assay solution was added and plates were incubated in the dark for 3 h. Relative cell viability was quantified by measuring the absorbance at 450 nm on a SpectraMax i3x multi-mode microplate reader. c Effect of vactosertib on TNF-α production by MM patient CD8 + T-cells. Cells (5000/well) were incubated with vactosertib at indicated concentrations for 24 h. Culture supernatant was centrifuged at 1000 × g for 5 min and cytokine level quantitated using the human TNF-α Quantikine ELISA kit. d Effect of vactosertib on IFN-γ production by patient CD8 + T-cells. Cells (5000/well) were incubated with vactosertib at indicated concentrations for 24 h. Culture supernatants were centrifuged at 3000 rpm × 5 min. Cytokine levels were quantitated using the human IFN-γ Quantikine duoSet ELISA kit. e Effect of vactosertib on autologous CD8 + T-cell cytotoxic activity. Shown is the effect of vactosertib and pomalidomide on autologous CD8 + T-cell activity against patient CD138 + cells. CD138 + cells (20,000/well) were treated as indicated for 8 h and then co-cultured with CD8 + T-cells (50,000/well) for 18 h. Cells were then stained to detect CD138 + cells followed by annexin-V and PI staining. Cells that gated positively for CD138 + , annexin-V and PI were quantitated by flow cytometry using FlowJo_10.8.1 software. In ( a – e ), values represent the average of triplicate measurements ( n = 3). Two-way ANOVA was conducted to investigate potential interactions between more than two variables ( a – e ). In panel ( a ), two asterisks (**) indicate a p -value ≤ 0.01. Source data are provided.

Article Snippet: CD138 + cells were isolated from the BMMC fraction using the EasySep TM human CD138 positive selection kit II (StemCell Technology, Cambridge, MA).

Techniques: XTT Assay, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture, Staining, Flow Cytometry, Software