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. 2024 Apr 11;143(15):1496-1512.
doi: 10.1182/blood.2023021671.

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome

Chella Krishna Vadivel  1 , Andreas Willerslev-Olsen  1 , Martin R J Namini  1 , Ziao Zeng  1 , Lang Yan  1 , Maria Danielsen  2 , Maria Gluud  1 , Emil M H Pallesen  1 , Karolina Wojewoda  3 , Amra Osmancevic  3 , Signe Hedebo  2 , Yun-Tsan Chang  4 , Lise M Lindahl  2 , Sergei B Koralov  5 , Larisa J Geskin  6 , Susan E Bates  7 , Lars Iversen  2 , Thomas Litman  1 , Rikke Bech  2 , Marion Wobser  8 , Emmanuella Guenova  4 , Maria R Kamstrup  9 , Niels Ødum  1 , Terkild B Buus  1
Affiliations

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome

Chella Krishna Vadivel et al. Blood. .

Abstract

Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus-specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

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Conflict of interest statement

Conflict-of-interest disclosure: C.K.V., N.Ø., and T.B.B. are listed as inventors on a patent application related to findings in this study (patent application EP23207309.8). N.Ø. has received consulting honoraria from Mindera Corp, Micreos Human Health, PS Consulting, and Almirall. E.M.H.P is currently employed at Novo Nordisk A/S. T.L. is funded by LEO Pharma. S.B.K.’s laboratory has previously received funding from Micreos, Dracen Pharmaceuticals, Kymera Therapeutics, and Bristol-Myers Squibb. R.B. has received research grants from Kyowa Kirin, Takeda, and Recordati. L.I. is also an employee at MC2 Therapeutics A/S. S.E.B. reports research funding to institution from Pfizer Inc., Merck, Amgen, RenovoRx, Agio, and the Pancreatic Cancer Action Network; payment or honoraria as an advisory board member for Servier, Elmedix, Pegascy, and Ipsen; and participation on a data safety monitoring board for Acrivon. K.W. has participated in advisory boards or lectures for Astra Zeneca, Galderma, and Kyowa Kirin. A.O. has participated in advisory boards or lectures for AbbVie, Celgene/Amgen, Eli Lilly, Novartis, Pfizer, Meda, UCB Pharma, Janssen Cilag, Allmiral, Kyowa Kirin, Sanofi, Bristol-Myers Squibb, Theracos, Recordati Rare Diseases, and Leo Pharma. M.W. received honoraria and participated in advisory boards of Takeda, Kyowa Kirin, Stemline Therapeutics, and Recordati Rare Diseases. The remaining authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
S aureus culture supernatants and SE induce drug resistance in malignant T cells. (A-C) Photographs of affected skin from 3 patients with SS before and after antibiotic treatment with ongoing cancer-directed treatments: (A) doxorubicin (SS1), (B) vorinostat (SS2), and (C) alitretinoin (SS3). (D) Flow cytometric plots showing apoptotic fraction of malignant T cells from SS17 PBMCs after 72 hours of treatment with 2 nM romidepsin and bacterial culture supernatant (sup) from S aureus cultured in the presence or absence of endolysin. The used S aureus strain was originally isolated from lesional skin of a different patient with SS. For control, tryptic soy broth medium (Ctrl medium) was added to the PBMC culture. (E) Western blot showing cleaved and uncleaved PARP expression after 24- and 48-hour treatment of SS4 PBMCs with either SE and/or romidepsin. GAPDH is used as a loading control. (F) Apoptotic fraction of malignant cells from PBMCs of 5 patients with SS (SS4, SS5, SS8, SS9, and SS12) after 72 and 144 hours treated with increasing concentrations of romidepsin in the presence (SE) or absence (PBS) of SE. Statistical significance was assessed by 2-way analysis of variance (ANOVA) followed by the Šídák multiple comparisons test. ∗∗P < .01; ∗∗∗P < .0005. (G) Apoptotic fraction of malignant cells from PBMCs of 2 patients with SS (SS8 and SS12) after a different 6 hours pulse, 16 hours chase in vitro treatment regimen with high concentrations of romidepsin in the presence (SE) or absence (PBS) of SE. (H-I) Representative flow cytometric plots (H; SS13) and quantification (I) of apoptotic fraction of malignant cells from PBMCs of 5 patients with SS (SS4, SS8, SS12, SS13, and SS15) after 72 hours of treatment with 2 nM romidepsin in presence of wild-type SEA or mutant SEA (SEAF47A/D227A). (J-K) Representative flow cytometric plots (J; SS15) and quantification (K) of apoptotic fraction of malignant cells from 4 patients with SS (SS4, SS12, SS13, and SS15) after 72 hours of treatment with 2 nM romidepsin in presence of SEA or SEA and a blocking anti-SEA antibody. For panels I-K, statistical significance was assessed by repeated measures 1-way ANOVA followed by Tukey multiple comparison test. ∗P < .05. Ctrl, control; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 2.
Figure 2.
SE largely overrides the transcriptional effect of romidepsin in malignant T cells. (A-B) Representative flow cytometric plots (A; SS8) and quantification (B) of percentage of viable malignant cells from PBMCs of 10 patients with SS (SS4, SS7, SS8, SS10, SS11, SS12, SS13, SS15, SS16, and SS17) treated with 2 nM romidepsin for 72 hours in the presence (SE) or absence (PBS) of SE. Red triangle shows the PBMCs of a patient with SS, which did not respond to romidepsin treatment. Statistical significance was assessed by ordinary 1-way ANOVA followed by Tukey multiple comparison test. ∗∗P < .01; ∗∗∗P < .0005. (C-E) Integrated uniform manifold approximation and projection (UMAP) from the 4 sample conditions (DMSO, romidepsin, SE, and SE + romidepsin) based on both mRNA and surface protein expression using totalVI colored by cell types (C), TCRβ CDR3 clonotype (D), and sample culture conditions (E; SS17). (F) Heatmap showing relative mean expression of malignant T cells after 36 hours treatment with romidepsin in the presence or absence of SE in SS17. Genes were selected based on differential expression between treatment and PBS control with a false discovery rate (FDR) <0.05 and a log2 fold change >0.5 or <−0.5 among any of the 3 treatments, yielding 574 genes in total. (G) Gene-set enrichment analysis (GSEA) of immune system pathways included in the Reactome database comparing 3 treatment conditions after 36 hours of culture (SS17): romidepsin (Ro), SE, and SE + romidepsin with PBS-treated control. Plot shows top 15 pathways from each comparison based on highest absolute normalized enrichment scores (NES) having a q-value <0.05. Other pathways are included in supplemental Figure 2E. (H) Transcription factor activity analysis using DoRothEA transcription factor signatures comparing 3 treatment conditions after 36 hours of culture (SS17): romidepsin, SE, and SE + romidepsin with PBS-treated control. Plot shows top 10 differentially activated transcription factors from each comparison based on highest absolute log2 fold change having an FDR <0.05. DMSO, dimethyl sulfoxide; mRNA, messenger RNA; NK cells, natural killer cells.
Figure 3.
Figure 3.
SE-mediated drug resistance of malignant T cells is not limited to romidepsin. Representative flow cytometric plots (SS12 [A,C]; SS4 [E]; SS17 [G]; SS7 [I]) and quantifications of percentage of viable malignant cells from PBMCs of patients with SS cultured for 72 hours in the presence or absence of SE and treated with 1 μM vorinostat (A-B) (n = 5 [SS5, SS7, SS10, SS12, and SS17]), 2 μM resminostat (C-D) (n = 3 [SS4, SS10, and SS12]), 100 to 400 nM doxorubicin (E-F) (n = 4 [SS4, SS7, SS10, and SS17]; due to fluorescent properties of doxorubicin being incompatible with PI staining, we used Mitotracker red CMXRos to mark viable cells), 10 to 50 μM etoposide (G-H) (n = 5 [SS7, SS8, SS10, SS11, and SS17]), or 50 nM bortezomib (I-J) (n = 4 [SS7, SS10, SS11, and SS12]). Statistical significance was assessed by repeated measures 1-way ANOVA followed by Tukey multiple comparisons test. ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005. DMSO, dimethyl sulfoxide; ns, not significant.
Figure 4.
Figure 4.
SE-induced drug resistance is mediated by TCR signaling via LCK-PKC-NF-κB. Representative flow cytometric plots (SS4 [A]; SS13 [C,E]) and quantifications of percentage of viable malignant cells from PBMCs of patients with SS treated with 2 nM romidepsin for 72 hours in the presence or absence of SE and 2 μM A-419259 (A-B) (Src inhibitor) (n = 8 [SS4, SS5, SS8, SS10, SS12, SS13, SS14, and SS15]), 100 nM dasatinib (C-D) (n = 6 [SS4, SS8, SS10, SS12, SS13, and SS15]), and 1.5 μM sotrastaurin (E-F) (PKC inhibitor) (n = 5 [SS4, SS10, SS12, SS13, and SS15]). Statistical significance was assessed by repeated measures 1-way ANOVA followed by Tukey multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. DMSO, dimethyl sulfoxide.
Figure 5.
Figure 5.
SE-induced drug resistance can be either JAK-dependent or -independent. (A) Heatmaps showing cytokines secreted by PBMCs derived from SS4 after 24, 48, and 72 hours of treatment with 2 nM romidepsin in the presence or absence of SE. (B) Heatmap showing cytokine expression across the cell types identified in the CITE-seq data set from SS17 after 36 hours of treatment with 2 nM romidepsin in the presence or absence of SE. Expression is calculated as log1p transformed unique transcripts per million log1p(CytokineUMIscelltypeTotalUMIssample/106). (C-D) Representative flow cytometric plots (C; SS13) and quantification (D) of percentage of viable malignant cells from PBMCs of 7 patients with SS (SS4, SS5, SS6, SS8, SS12, SS13, and SS14) treated with 2 nM romidepsin for 72 hours in the presence or absence of cytokines (IL-2, IL-4, IL-7, and IL-15). Statistical significance was assessed by ordinary 1-way ANOVA followed by Tukey multiple comparison test. ∗∗∗∗P < .0001. (E-H) Representative flow cytometric plots (SS8 [E]; SS10 [G]) and quantifications of percentage of viable malignant cells from PBMCs of patients with SS treated with 2 nM romidepsin for 72 hours in the presence or absence of SE and JAK inhibitor (1 μM tofacitinib) of patients exhibiting JAK-independent (E-F) (n = 3 [SS8, SS12, and SS13]) or JAK-dependent (G-H) (n = 2 [SS4 and SS10]) resistance being induced by the presence of SE. (E) PBMCs from SS8 treated in the presence of cytokines (IL-2, IL-4, IL-7, and IL-15) as well as SRC inhibitor (2 μM A-419259). DMSO, dimethyl sulfoxide; NK cells, natural killer cells.
Figure 5.
Figure 5.
SE-induced drug resistance can be either JAK-dependent or -independent. (A) Heatmaps showing cytokines secreted by PBMCs derived from SS4 after 24, 48, and 72 hours of treatment with 2 nM romidepsin in the presence or absence of SE. (B) Heatmap showing cytokine expression across the cell types identified in the CITE-seq data set from SS17 after 36 hours of treatment with 2 nM romidepsin in the presence or absence of SE. Expression is calculated as log1p transformed unique transcripts per million log1p(CytokineUMIscelltypeTotalUMIssample/106). (C-D) Representative flow cytometric plots (C; SS13) and quantification (D) of percentage of viable malignant cells from PBMCs of 7 patients with SS (SS4, SS5, SS6, SS8, SS12, SS13, and SS14) treated with 2 nM romidepsin for 72 hours in the presence or absence of cytokines (IL-2, IL-4, IL-7, and IL-15). Statistical significance was assessed by ordinary 1-way ANOVA followed by Tukey multiple comparison test. ∗∗∗∗P < .0001. (E-H) Representative flow cytometric plots (SS8 [E]; SS10 [G]) and quantifications of percentage of viable malignant cells from PBMCs of patients with SS treated with 2 nM romidepsin for 72 hours in the presence or absence of SE and JAK inhibitor (1 μM tofacitinib) of patients exhibiting JAK-independent (E-F) (n = 3 [SS8, SS12, and SS13]) or JAK-dependent (G-H) (n = 2 [SS4 and SS10]) resistance being induced by the presence of SE. (E) PBMCs from SS8 treated in the presence of cytokines (IL-2, IL-4, IL-7, and IL-15) as well as SRC inhibitor (2 μM A-419259). DMSO, dimethyl sulfoxide; NK cells, natural killer cells.
Figure 6.
Figure 6.
SE induce drug resistance in purified malignant T-cell cultures via JAK-dependent or -independent mechanisms. (A) Flow cytometric plot showing percentage of malignant T cells before and after sorting of PBMCs from SS4. (B-C) Flow cytometric plots showing percentage of viable malignant cells from sorted malignant T cells treated with 2 nM romidepsin for 72 hours in the presence or absence of SE and JAK inhibitor (1 μM tofacitinib) of patient exhibiting JAK-independent (SS13) (B) and JAK-dependent (SS4) (C) resistance being induced by the presence of SE. DMSO, dimethyl sulfoxide.
Figure 7.
Figure 7.
S aureus induces drug resistance in malignant T cells through direct and indirect TCR activation.S aureus can induce drug resistance in malignant T cells through secretion of SE using 2 distinct pathways: (1) direct TCR-dependent, JAK-independent pathway mediated through PKC-dependent NF-κB signaling (upper part), and (2) an indirect JAK-dependent pathway, which relies on SE-induced cytokine production in bystander T cells or the malignant cells themselves in an auto/paracrine fashion (lower part).
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