Presentation_1_Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.pptx

IntroductionBCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells.MethodsCo-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB.ResultsCFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins.ConclusionStromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.</p

Additional file 1 of “What I wanted to do was build myself back up and prepare”: qualitative findings from the PERCEPT trial of prehabilitation during autologous stem cell transplantation in myeloma

Supplementary Material

The Effects of CAMPATH-1H on Cell Viability Do Not Correlate to the CD52 Density on the Cell Surface

<div><p>Graft versus host disease (GvHD) is one of the main complications after hematological stem cell transplantation (HSCT). CAMPATH-1H is used in the pre-transplant conditioning regimen to effectively reduce GvHD by targeting CD52 antigens on T cells resulting in their depletion. Information regarding CD52 expression and the effects of CAMPATH-1H on immune cells is scant and limited to peripheral blood (PB) T and B cells. To date, the effects of CAMPATH-1H on cord blood (CB) cells has not been studied. Here we aimed to analyze CD52 expression and the effects of CAMPATH-1H on fresh or frozen, resting or activated, PB mononuclear cells (PBMC) and CB mononuclear cells (CBMC). In resting state, CD52 expression was higher in CB than PB T cell subsets (653.66±26.68 vs 453.32±19.2) and B cells (622.2±20.65 vs 612.0±9.101) except for natural killer (NK) cells where CD52 levels were higher in PB (421.0±9.857) than CB (334.3±9.559). In contrast, CD52 levels were comparable across all cell types after activation. CAMPATH-1H depleted resting cells more effectively than activated cells with approximately 80–95% of apoptosis observed with low levels of necrosis. There was no direct correlation between cell surface CD52 density and depleting effects of CAMPATH-1H. In addition, no difference in cell viability was noted when different concentrations of CAMPATH-1H were used. CD52 was not expressed on HSC but began to be expressed as the cells differentiate, implying that CAMPATH-1H could potentially affect HSC differentiation and proliferation. Our study provides insightful information, which contributes to the better understanding in the use of CAMPATH-1H as part of the conditioning regime in HSCT.</p></div

Viability of PB and CB naïve and memory T cells after treatment with CAMPATH.

<p><b>A, C, E, and G</b> show the percentage of apoptotic cells in resting/activated naïve CD4, naïve CD8, memory CD4, and memory CD8 T cells respectively. <b>B, D, F, and H</b> show the percentage of necrosis in resting/activated naïve CD4, naïve CD8, memory CD4, and memory CD8 T cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005. Significant differences were observed between resting and activated naïve and memory T cells across all fresh samples with p value<0.005 (n = 5).</p

CD52 expression and effects of CAMPATH on HSC and HSPC.

<p><b>A and B</b> show the level of CD52 expression in CD45^high/low lymphoid and myeloid progenitors in cultures of CB and PB HSC respectively. <b>C and D</b> show the number of colony from CB SC and mPB SC respectively at day 14. <b>E and F</b> show the percentage of NK cells formed during the 35 days of culture of CB SC and mPB SC respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 3).</p

Viability of PB and CB Treg cells after treatment with CAMPATH.

<p><b>A</b> shows the percentage of apoptotic cells in resting/activated Treg cells. <b>B</b> shows the percentage of necrosis in resting/activated Treg cells. *represents p value<0.05, **represents p value<0.002, ***represents p value<0.005. Significant differences were observed between resting and activated Treg cells across all fresh samples with p value<0.005 (n = 5).</p

CD52 expression on different CB and PB cell subsets.

<p><b>A and C</b> show the level of CD52 expression in fresh and frozen resting immune cells respectively. <b>B and D</b> show CD52 expression in fresh and frozen activated immune cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 5).</p

Viability of activated PB and CB immune cells after treatment with CAMPATH-1H in the presence or absence of complement.

<p><b>A, C, E, G, and I</b> show the percentage of apoptotic cells in naïve T cells, memory T cells, Treg cells, NK cells, and NKT cells respectively. <b>B, D, F, H, and J</b> show the percentage of necrosis in naïve T cells, memory T cells, Treg cells, NK cells, and NKT cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 4/5).</p

5T2MM bearing mice have increased bone loss in the lumbar vertebrae and calvariae.

<p>A. Photomicrographs of the vertebrae from naïve and 5T2MM bearing mice. Tumour infiltration is identified with black arrows. B. The proportion of trabecular bone area in the vertebrae from naïve and 5T2MM-bearing mice. C. Photomicrographs of the calvariae of naïve and 5T2MM bearing mice. Medullary spaces are identified with black arrows, and OC with yellow arrows. Non tumour-containing spaces are shown. D. The proportion of medullary area as a percentage of total tissue area in naïve and 5T2MM-bearing mice. E. Distribution of medullary spaces in naïve and 5T2MM-bearing mice, showing significant differences in distribution (KS test, p<0.02). Chi-square analysis demonstrated that 5T2MM-bearing mice had significantly fewer, smaller spaces and more, larger spaces than naïve mice (p<0.05). Data = mean± S.E.M. (B and D).</p

Serum concentrations of sRANKL and OPG are abnormally regulated in mice bearing 5T2MM and 5T33MM cells, respectively.

<p>Serum concentrations of sRANKL and OPG are abnormally regulated in mice bearing 5T2MM and 5T33MM cells, respectively. A. Serum concentrations of sRANKL in naive mice and mice bearing 5T2MM or 5T33MM cells. B. Serum concentrations of OPG in naive animals and animals bearing 5T2MM or 5T33MM cells. Statistical analysis by Mann-Whitney U test. Data = mean± S.E.M.</p