Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from peripheral TCL patients treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in interferon signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation.

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2024
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Supplementary data

Table S1

Table S1. Details of primary antibodies used in Western blots in this study (Figures 1,2,4,6 & S1).

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Table S2

Table S2. Details of TaqMan primers used in qPCR assays in this study (Figures 2,5 & S9).

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Table S3

Table S3. Generation of CRISPR Knockout (KO) and CRISPR Activation (CRISPRa) Cell Line Clones

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Figure S1

Figure S1. Additional Western blots showing different CRISPR clones generated from each construct. (Refers to Figure 1)

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Figure S2

Figure S2. Additional viability and cytokine data from each TCL CRISPR clone experiment. (Refers to Figure 1)

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Figure S3

Figure S3. Additional EL4-C8KO Western blot and lytic cell death data. (Refers to Figure1)

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Figure S4

Figure S4. Changes in DNA methylation levels after decitabine (DAC) treatment of human TCL cell lines. (Refers to Figure 2)

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Figure S5

Figure S5. Combination viability assay raw data. (Refers to Figure 3)

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Figure S6

Figure S6. Additional Karpas-299 cytokine data. (Refers to Figure 4)

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Figure S7

Figure S7. Additional in vivo PD data. (Refers to Figure 5)

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Figure S8

Figure S8. Additional EL4 & EL4-C8KO model in vivo efficacy data. (Refers to Figure 6)

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Figure S9

Figure S9. Analysis of tumor and plasma samples from EL4-Parental and EL4-C8KO syngeneic model PD studies (Refers to Figure 6)

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Figure S10

Figure S10. Impact of tolinapant and decitabine on induction of immunogenic cell death in TCL. (Refers to Discussion)

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Figure S11

Uncropped Western blots from Figure 2A

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