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Therapeutic synergy between tigecycline and venetoclax in a preclinical model of MYC/BCL2 double-hit B cell lymphoma

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Science Translational Medicine  31 Jan 2018:
Vol. 10, Issue 426, eaan8723
DOI: 10.1126/scitranslmed.aan8723
  • Fig. 1 Blockade of tigecycline-induced cell death by BCL2 in Eμ-myc lymphomas and its restoration by venetoclax.

    Two mouse Eμ-myc lymphomas, one wild type (p53 wt) and one null (p53 null), for the Trp53 locus were infected with a recombinant retrovirus encoding human BCL2 or with the corresponding empty vector (EV). After selection, the infected cells were treated with tigecycline and/or venetoclax at the indicated concentrations. Total and viable cell counts were determined by Trypan blue exclusion 48 hours after addition of the drugs to the culture medium. (A) The viability of cells expressing BCL2 or EV is presented as the percentage of live relative to total cells (live + dead) in each culture. (B) The proliferative index of cells expressing BCL2 or EV is presented as the increase in viable cell counts over 48 hours, normalized to the corresponding increase in parallel untreated cultures (4 population doublings for the p53 wt cells with either EV or BCL2; 3.5 and 2 doublings for the p53 null cells with EV and BCL2, respectively). (C) Cell viability of Eμ-myc/BCL2 lymphomas in the presence or absence of venetoclax. (D) Cell viability of Eμ-myc/BCL2 lymphomas in the presence or absence of tigecycline and/or venetoclax. All data points represent the average ± SD from three independent experiments.

  • Fig. 2 Induction of cell death by tigecycline and venetoclax in human MYC/BCL2 DHL.

    (A and B) MYC and BCL2 mRNAs were quantified by RT-PCR (reverse transcription polymerase chain reaction) with normalization to the RPLP0 mRNA (n = 3 technical replicates for each group) (A) or immunoblotting (B) in the indicated cell lines. The expression data shown here are consistent with the known status of MYC or BCL2 translocations in those cell lines, as indicated at the bottom. Note that in SU-DHL-6 cells, somatic mutations in BCL2 impair its recognition by the OP60 antibody (n.a.) (65), but the protein is readily detected by antibody E17. (C) Cultures were treated with tigecycline and/or venetoclax at the indicated concentrations, and cell viability was determined as in Fig. 1.

  • Fig. 3 Therapeutic activity of tigecycline and venetoclax in DHL-xenografted mice.

    The human cell lines SU-DHL-6, DOHH-2, OCI-LY8 (all with MYC and BCL2 translocations), or OCI-LY7 (MYC translocation only) were xenografted subcutaneously in CD1-nude mice. After about 2 weeks, tumor-bearing animals were randomized and treated with the indicated doses of tigecycline and/or venetoclax over a period of 12 days. Tumor volumes were measured at the indicated time points after treatment. For DOHH-2, OCI-LY8, and OCI-LY7, tumor growth is shown at every time point until termination. For SU-DHL6 tumors, only days 0 to 19 are included in the graph: Of the eight animals treated with tigecycline + venetoclax, six reached the 120-day end point tumor-free, and two developed tumors and were sacrificed at day 50 (table S1). Error bars represent SD; #P = 5 × 10−3, +P < 5 × 10−4, and *P < 10−5; ns, not significant.

  • Fig. 4 Therapeutic activity of tigecycline and venetoclax in a DHL patient-derived xenograft.

    The patient-derived xenograft line DFBL-69487-V3-mCLP, expressing luciferase, was expanded by intravenous injection in NSG mice. Fifteen days after seeding (day 0), tumor development was monitored by whole-body imaging: Animals were randomized (five animals per group), subjected to treatment with tigecycline (75 mg/kg) and/or venetoclax (50 mg/kg) over a period of 12 days (days 1 to 12), and monitored by whole-body imaging at the indicated time points (before, during, and after treatment). (A) Tumor progression in the indicated groups of animals was followed by radiant efficiency, quantified over both femurs. (B) IVIS imaging (ventral and lateral) of all mice in the control (untreated) and tigecycline + venetoclax (tig + ven) groups, shown at day 11. Other groups and time points are shown in fig. S7A. Arrowheads point to the accumulation of tumor cells in the femurs (f.) and vertebrae (v.). The red circles and rectangles appearing over the photographs define the areas that were used for quantification of luminescence over the femur and the corresponding values, respectively. All values are provided in table S1.

  • Table 1 Summary of all treatments performed on mice with SU-DHL-6–derived tumors.

    n mice (died ≤1 week) refer to the total numbers of mice treated and, in parentheses, those that died within the first week. The causes for these early deaths remain unclear and may be due to the overall experimental burden imposed on the animals; surviving animals showed no signs of distress. Regression: Percentages of animals scored as showing tumor regression at the indicated time points, with the scored/total numbers in parentheses. At day 19, partial and complete regressions were defined as residual tumor volumes ≤50% and ≤10% of the initial volume, respectively. Animals showing complete regression at day 19 were followed until day 120 and sacrificed when tumors reached a diameter of 2 cm. Animals reaching the end point of 120 days had no detectable residual tumor. Compacted liver: “Yes” indicates that the postmortem examination revealed a compacted liver lobule morphology.

    Tigecycline,
    mg/kg
    Venetoclax,
    mg/kg
    n mice
    (died ≤1 week)
    Regression: % tumor-free (n)Compacted
    liver
    Day 19: PartialDay 19: CompleteDay 120
    0.9% NaCl14
    Vehicle13
    757Yes
    10012Yes
    2514
    506
    1007
    1001007 (3)75% (3/4)75% (3/4)Yes
    100506100% (6/6)100% (6/6)Yes
    1005010 (2)12.5% (1/8)87.5% (7/8)50% (4/8)Yes
    10025757% (4/7)Yes
    7510010 (5)20% (1/5)80% (4/5)60% (3/5)Yes
    751008 (3)100% (5/5)60% (3/5)Yes
    7550812.5% (1/8)87.5% (7/8)75% (6/8)Yes
    50507100% (7/8)42% (3/7)
    25100728% (2/7)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/426/eaan8723/DC1

    Fig. S1. Response of mouse Eμ-myc/BCL2 lymphomas to tigecycline and venetoclax.

    Fig. S2. Synergistic effects of tigecycline and venetoclax against human DHL cell lines.

    Fig. S3. Similar effects of tigecycline, doxycycline, and tetracycline in DHL cells.

    Fig. S4. Short-term effects of tigecycline and venetoclax in SU-DHL-6 tumors.

    Fig. S5. Reactive liver lesions caused by intraperitoneal delivery of tigecycline.

    Fig. S6. Toxicology of tigecycline and venetoclax drug combination studies in vivo.

    Fig. S7. Whole-body imaging of PDX-derived tumors treated with tigecycline and/or venetoclax.

    Fig. S8. Kaplan-Meier representation of disease-free survival in PDX-recipient mice.

    Fig. S9. Effect of rituximab with tigecycline and/or venetoclax on DHL xenografts.

    Fig. S10. Whole-body imaging of PDX-derived tumors treated with rituximab, tigecycline, and/or venetoclax.

    Table S1. Primary data.

  • Supplementary Material for:

    Therapeutic synergy between tigecycline and venetoclax in a preclinical model of MYC/BCL2 double-hit B cell lymphoma

    Micol Ravà, Aleco D'Andrea, Paola Nicoli, Ilaria Gritti, Giulio Donati, Mirko Doni, Marco Giorgio, Daniela Olivero, Bruno Amati*

    *Corresponding author. Email: bruno.amati{at}ieo.it

    Published 31 January 2018, Sci. Transl. Med. 10, eaan8723 (2018)
    DOI: 10.1126/scitranslmed.aan8723

    This PDF file includes:

    • Fig. S1. Response of mouse Eμ-myc/BCL2 lymphomas to tigecycline and venetoclax.
    • Fig. S2. Synergistic effects of tigecycline and venetoclax against human DHL cell lines.
    • Fig. S3. Similar effects of tigecycline, doxycycline, and tetracycline in DHL cells.
    • Fig. S4. Short-term effects of tigecycline and venetoclax in SU-DHL-6 tumors.
    • Fig. S5. Reactive liver lesions caused by intraperitoneal delivery of tigecycline.
    • Fig. S6. Toxicology of tigecycline and venetoclax drug combination studies in vivo.
    • Fig. S7. Whole-body imaging of PDX-derived tumors treated with tigecycline and/or venetoclax.
    • Fig. S8. Kaplan-Meier representation of disease-free survival in PDX-recipient mice.
    • Fig. S9. Effect of rituximab with tigecycline and/or venetoclax on DHL xenografts.
    • Fig. S10. Whole-body imaging of PDX-derived tumors treated with rituximab, tigecycline, and/or venetoclax.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

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