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Oncolytic immunotherapy and bortezomib synergy improves survival of refractory multiple myeloma in a preclinical model

Chandini M. Thirukkumaran, Zhong Qiao Shi, Gerard J. Nuovo, Joanne Luider, Karen A. Kopciuk, Yuan Dong, Ahmed A. Mostafa, Satbir Thakur, Kathy Gratton, Ailian Yang, Alex C. Chin, Matt C. Coffey, Victor H. Jimenez-Zepeda, Douglas Stewart, Marta Chesi, P. Leif Bergsagel and Don Morris

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    In vitro synergy of reovirus with BTZ in oncolysis of RPMI 8226, KMS11, and OPM2 human myeloma cell lines. (A) Viability of human myeloma cells following single or dual treatment. RPMI 8226, KMS11, and OPM2 cells were treated with constant ratios of ED50 values generated for reovirus (2.31, 13.25, and 63.05 MOI, respectively) and BTZ (2.8, 4.76, and 0.87 nM, respectively). Cell viability was assessed at 48 hours via the WST assay. N = 4 independent experiments. *P < .05, Conover’s test. (B) Viral progeny production in RPMI8226, KMS11, and OPM2 following treatment with LV alone or LV in combination with BTZ. Myeloma cells in 24-well plates were infected with ED50 values of LV or a combination of ED50 values of LV and BTZ (as in panel A). Samples were collected at 12-hour time points up to 72 hours; virus yields were determined by plaque titration on L929 cells and represented as log plaque-forming unit per milliliter. The linear rates of viral progeny production in the 2 groups were similar within the 3 cell lines, but the rate in the RPMI8226 cell line was nearly triple that of OPM2. N = 3, 3 independent experiments. DV accomplished through ultraviolet-inactivation.25

  • Figure 2.

    Early tumor microenvironment events following RV + BTZ combination therapy in vivo. (A) Schematic representation of treatment time line in mice. (B) To evaluate early tumor microenvironment events, mice were euthanized after 4 days of treatment; their spleens and BM were then formalin fixed and paraffin embedded. Serial sections were stained and scored for various markers (supplemental Table 1) per previously published IHC protocols.54-56 Optimal conditions were determined using various myeloma cell lines with different sensitivities infected with RV, and included a dilution of 1:1000 and a rabbit/anti-goat secondary antibody (ABCAM, 1:500 dilution). Myeloma tumor burden in the treated cohorts for Figures 2B-F and 3-5 are given in Table 3. Reoviral capsid protein was detected within myeloma cells in scattered foci in mice treated only with LV (arrows), whereas many more viral positive myeloma cells were seen in the spleen after live virus and BTZ treatment (magnification ×200 = ×10 [ocular lens] and ×20 [objective lens]). (C) LV + BTZ combination therapy leads to significantly higher RV protein production. Bar diagrams representing quantified LV RNA and protein in BM and spleen myeloma tumor. N = 3; ***P < .001, Conover’s test. (D) Splenic myeloma caspase 3 expression images of IHC sections. Note the low activated caspase-3 expression in the mice treated with the VC or DV. Foci of myeloma cells positive for caspase-3 were seen in the spleen of the mice treated with LV alone, whereas there was a diffuse and strong increase in caspase-3 expression in the mice treated with LV and BTZ (magnification ×200 = ×10 and ×20). (E) Significantly higher caspase 3 is expressed in MM tumor of LV + BTZ combination therapy. Bar diagrams representing quantified caspase 3. N = 3; ***P < .001, **P < .01, *P < .05, Conover’s test. (F) Coexpression of CD138 and caspase 3 in spleen myeloma cells. Coexpression of caspase 3 in CD138+ myeloma cells (right, fluorescent yellow) in RV + BTZ–treated spleens confirms that it is primarily myeloma cells that undergo apoptosis (magnification ×400 = ×10 and ×40). (B,D) Camera: Ventana Vias Zeiss Axio; acquisition software: Nuance. Composite figure via Adobe Photoshop CS4. PFU, plaque-forming unit; TFC, tumor-free control.

  • Figure 3.

    Early tumor microenvironment events indicate that RV + BTZ combination therapy leads to significantly enhanced immune activation. (A) IHC staining of CD3 in spleens of mice. Note the rare tumor associated CD3+ cells in the mice treated with the VC compared with the strong infiltration by such cells in the tumor after treatment with LV and BTZ (magnification ×200 = ×10 and ×20). (B) Bar plots representing quantified CD3+ cells in BM and spleen. N = 3; **P < .01, *P < .05, Conover’s test. (C) Bar plots representing quantified CD117+ IL22+ NK cells in BM. N = 3; **P < .01, *P < .05, Conover’s test. (D) IHC sections of mouse spleens stained for NK cells. IHC staining of CD117+ IL22+ NK cells in spleens of mice. Note the rare NK cell distribution in LV-treated spleens, robust expression of NK cells in LV + BTZ–treated spleens, and red pulp areas of mouse spleen (magnification ×200 = ×10 and ×20). (A,D) Camera: Ventana Vias Zeiss Axio; acquisition software: Nuance. Composite figure via Adobe Photoshop CS4.

  • Figure 4.

    RV + BTZ combination therapy upregulates immune check point expression in MM tumor as early as 4 days posttreatment. (A) IHC staining images of spleen myeloma PD-L1 in mice. Note the rare expression of PD-L1 in the tumors of mice treated with the VC as compared with the strong expression of this checkpoint protein in the tumor after treatment with live virus and BTZ (magnification ×200 = ×10 and ×20; camera: Ventana Vias Zeiss Axio; acquisition software: Nuance; composite figure via Adobe Photoshop CS4). Bar plots representing quantified PD-L1 (B), PD-L2 (C), and IDO expression (D) in MM cells in BM and spleen. N = 3; ***P < .001, **P < .01, *P < .05, Conover’s test.

  • Figure 5.

    Dual treatment of RV + BTZ upregulates TME-associated M2 type macrophages in mouse BM. (A) Images depicting IHC staining for IL-10 in mouse BM. Note the low IL-10 expression in the mice treated with VC or LV alone compared with the strong expression of this cytokine in the BM after treatment with LV and BTZ (magnification ×200 = ×10 and ×20; camera: Ventana Vias Zeiss Axio; acquisition software: Nuance; composite figure via Adobe Photoshop CS4). (B) Bar plots representing quantified BM F4/80+/CD31+ macrophages (left) and IL-10–secreting M2 macrophages (right). N = 3; ***P < .001, **P < .01, *P < .05, Conover’s test. (C) Images depicting CD11b+Gr1+ (classic markers of MDSC) staining in mouse spleen TME (magnification ×200 = ×10 and ×20). At early stages (day 4) posttreatment, there is no difference in CD11b+Gr1+ MDSCs between vehicle control and the treated samples.

  • Figure 6.

    RV + BTZ treatment leads to significant reductions in MM tumor burden and superior OS. C57BL/6 wild-type recipient mice were injected with Vk12598 myeloma cells (8 × 105) IV and treated per the schematic in Figure 2A. Mice were bled weekly posttreatment and serum paraprotein was evaluated via high-resolution serum electrophoresis. (A) Left: M-spike changes in mice posttreatment from week W1 through W4. GEE analysis up to W3 indicated that cohorts of LV, LV + BTZ, DV + BTZ, and BTZ were different from the VC group at W2 and W3, but not at W1 (P < .05). DV vs VC showed no differences at all time points. GEE trend analysis of W1 through W4 data also indicated treatment groups were different (P < .05) from the VC group at W2, W3, and W4, but not at W1. Center and left: Mean M-spike data at weeks 3 and 4, respectively. (B) Kaplan-Meier survival plots of mice posttreatment. P < .00001, log-rank test.

  • Figure 7.

    RV + BTZ treatment leads to significant immune modulation, memory cell generation, and reverses myeloma-induced immune suppression. Mouse spleens and BM of the long-term survival experiment (Figure 6B) were harvested whenever they depicted extreme morbidity. Cells were immunophenotyped for CD8+, CD4+ T cells, NKT, and Treg and T- effector memory cells. Dot plots representing CD8+ T cell (A), CD4+ T cell (B), CD3+CD49b+ NKG2D+NKT cell (C), and Foxp3+CD4+ Treg cell (D) percent of lymphocytes. N = 6; ***P < .001, **P < .01, *P < .05, Conover’s test. (E-G) Dot plots representing TAMs and MDSCs. (E) CD45+CD11b+Ly6Clow Ly6G TAMs. (F) CD45+CD11b+Ly6G+ PMNC MDSCs. (G) CD45+CD11b+Ly6C+ monocytic origin MDSCs. N = 6; ***P < .001, **P < .01, *P < .05, Conover’s test. (H) Myeloma tumor burden of treated cohorts assessed by M-spike. N = 6; ***P < .001, **P < .01, *P < .05, Conover’s test. Bar plots depicting BM (I) and splenic (J) memory T cells analyzed by flow cytometry. N = 4; ***P < .001, **P < .01, *P < .05, Conover’s test. PMNC, polymorphonuclear.

Tables

  • Table 1.

    RV and BTZ combination therapy ED50 values

    Cell lineRV MOIBTZ, nM
    RPMI 82262.312.80
    KMS1113.254.76
    OPM263.050.87
  • Table 2.

    RV and BTZ combination therapy CI values

    Cell lineDose 1: ED50/8Dose 2: ED50/4Dose 3: ED50/2Dose 4: ED50Dose 5: 2 × ED50
    RPMI82263.31 ± 1.692.01 ± 0.672.30 ± 0.550.70 ± 0.010.43 ± 0.01
    KMS110.16 ± 0.700.05 ± 0.010.06 ± 0.010.10 ± 0.010.17 ± 0.03
    OPM20.12 ± 0.010.20 ± 0.020.29 ± 0.020.38 ± 0.040.30 ± 0.02
    • CI values for RV and BTZ were generated using Calcusyn software. Synergism was determined per Chou-Talalay method; bold type indicates CI <1, denoting a synergistic response, CI >1 denotes an antagonistic response, and CI = 1 denotes an additive response.

  • Table 3.

    Myeloma tumor burden (mean M-spike) of mice at onset of treatment and percent infiltration of tumor in mouse spleen and BM 4 d posttreatment

    M-spike (SE), g/LSpleen % tumor infiltration (SE)BM % tumor infiltration (SE)
    VC6.15 (2.46)69.5 (1.5)61.5 (4.5)
    DV7.33 (2.78)81 (5)65 (12)
    DV + BTZ4.85 (3.65)50 (9)46.5 (5.5)
    BTZ4.37 (4.27)41 (28)32 (5)
    LV4.54 (3.05)48.5 0.5)48.5 (6.5)
    LV + BTZ13.14 (7.38)57.5 (8.5)68.5 (8.5)
    • SE, standard error.

  • Table 4.

    Cytokine and immune modulatory molecule upregulation in mouse myeloma TEM following RV and BTZ combination treatment

    SampleIFN-β, no. + MNC/×200 (SD)IFN-γ, no. + MNC/×200 (SD)MHC1, no. + MNC/×200 (SD)TAP1, no. + MNC/×200 (SD)TLR3, no. + MNC/×200 (SD)NF-κB, no. + MNC/×200 (SD)
    Tumor-free control spleen0012.3 (4.4)2.0 (0.7)01.1 (0.4)
    Vehicle control myeloma spleen7.1* (1.3)15.2 (5.5)19.5 (4.0)4.1 (1.1)13.5 (2.3)12.2 (3.3)
    DV myeloma spleen4.9 (2.0)11.9 (5.9)27.4 (7.9)12.2 (4.7)20.1 (6.3)32.9 (6.0)
    BTZ myeloma spleen15.0 (6.6)23.2 (8.2)29.0 (7.7)5.0 (2.8)34.3 (7.7)33.9 (5.7)
    DV + BTZ myeloma spleen18.1 (5.8)28.9 (9.0)25.2 (8.2)15.9 (3.3)27.9 (9.2)28.9 (7.1)
    LV myeloma spleen (low productive infection)14.1 (4.9)18.8 (4.3)111.9 (9.7)48.9 (7.7)109.4 (9.2)69.5 (5.9)
    LV + BTZ myeloma spleen (productive infection)83.2 (8.95)76.1 (7.9)218.15 (17.1)91.3 (9.65)116.0 (9.25)76.35 (7.1)
    • SD, standard deviation; MNC, mononuclear cell.

    • * All cell counts were done at the interface of myeloma cells and normal spleen and represent the number of positive cells at ×200. N = 2, mean of 10 fields (SD).