Small-Molecule Chemotherapeutic Drugs Reactivate HIV-1 via Non-Canonical Pathways and Modulate CD8 T cell response

Transcription

1 Small-Molecule Chemotherapeutic Drugs Reactivate HIV-1 via Non-Canonical Pathways and Modulate CD8 T cell response ~ Dr. Isa Munoz-Arias, Ph.D Lab of Dr. Timothy Henrich, M.D UCSF Department of Experimental Medicine IAS HIV Cure & Cancer Forum Paris, France

2 Background Drugs that can reactivate the latent reservoir without global cell activation while enhancing HIV-specific immune responses are an HIV cure priority. Goal of the project: Screen FDA approved chemotherapeutic drugs for the ability to reactivate HIV and enhance anti-hiv immunity, and study successful candidates for mechanism.

3 Name Chemo Drug Screen for HIV Reactivation JLATs and Patient Derived CD4 T cells Inhibition Targets RTK inhibitor: PDGFR, VEGFR, ckit, FLT3, Reactivation in JLATS, 5A8s Sunitinib FGFR1 Yes Yes Sorafenib TK Inhibitor: PDGFR, VEGFR/FLK1, RAF, B- RAF, FLT3 No Yes Bortezomib Proteosome inhibitor No Yes Dasatinib TK inhibitor: Abl, Src, ckit No No Imatinib TK inhibitor: v-abl, c-kit, PDGFR No No Bosutinib TK inhibitor: Src/Abl No No Nilotinib TK inhibitor: BCR-Abl No No Gefitinib TK inhibitor: EGFR No No Erlotinib TK inhibitor: EGFR No No Doxorubicin (Doxil) Anthracyclin antibiotic: Topo Isomerase II No No Lestaurtinib FLT-3, JK2, TrkA No Cabozantinib VEGFR-2, FLT-3 No Ponatinib VEGFR-2 No Patient CD4

4 Target Drug Background Bortezomib is a proteosome inhibitor used to treat multiple myeloma and mantle cell lymphoma Sorafenib is a VEGF, PDGF inhibitor used for hepatocellular, liver, and thyroid carcinomas Sunitinib is anti-angiogenic and immunomodulating chemo drug used against metastatic renal and hepatocellular carcinomas

5 Methods HIV + Patient Blood lymphocytes 48 hrs RNA/DNA Extraction From Cells qpcr Cell associated HIV Gag RNA /CCR5 DNA Per 1x10^6 cells 0.5-3x10 6 CD4 T cells/well Chemo-drugs at (4 16 x IC50)

6 Bortezomib and Sunitinib Enhance HIV-1 Reactivation Donor 33 Donor 41 Donor 38 Tim and Emily s Data Suppressed Patient Blood derived CD4 T cells

7 Bortezomib Enhances HIV Reactivation from Suppressed and Elite Patient Samples Bortezomib x IC50 *IC50 = 100nM For Proteosome

8 Sorafenib Enhances HIV Reactivation from Suppressed Patient Samples Sorafenib x IC50 *IC50 = 90nM For VEGF

9 Sunitinib Enhances HIV Reactivation from Suppressed and Elite Patient Samples Sunitinib x IC50 *IC50 = 80nM For VEGF

10 Surface Activation Markers Bortezomib CD69- no change CD25- no change HLADR- no change Sorafenib CD69- no change CD25- no change HLADR- no change Sunitinib CD69- Increase CD25- no change HLADR- no change

11 Question Is Sunitinib as efficient as Bryostatin and Romodepsin at reactivating HIV from suppressed patient samples?

12 Sunitinib Reactivates More HIV than Bryostatin and Romodepsin from Suppressed Patient Samples (N=3)

13 Is Sunitinib inducing reactivation via the canonical pathway? NFAT 1 NFAT 2 HIV-1 LTR CEβpβ NFƙB 1 NFƙB 2 AP1 SP1 SP1 SP1 TATA NFAT 2 NFƙB- canonical 3 NFAT- canonical 1 AP1 3 SP1 4 CEβP-Only 1 functional site

14 Sunitinib does not induce transcription of NFƙB or NFAT targets by quantitative PCR Treatment NF-ƙB IKK- TNF IFN IL10 IL6 Untreated >1 Sunitinib >1 CD3/CD Results are from 1 HIV + patient (patient #39) out of 4 tested Sunitinib = 16X IC50

15 Question Does Sunitinib enhance NFƙB activation in a reporter cell line?

16 GFP

17 Sunitinib Does Not Increase NFƙB Dependent DsRed Expression in 5A8s Untreated CD3/CD28/IL2 Sunitinib 16x IC50 NFƙB-DsRed CD69

18 Question Does Sunitinib enhance CD8 T cell degranulation in patient samples treated with pooled Gag peptides?

19 Methods HIV + Patient PBMC 1x10^6 PBMC /well Sunitinib at (4 8x IC50) Overlapping Gag Peptides (0.4ug/mL) 5 days Stained for CD107a Treated with Golgi-Stop FACS analysis

20 Sunitinib Increases CD8 T cell Degranulation as Measured by CD107a Expression No Drug Sunitinib 8x Sunitinib 16x CD3/CD28/IL2 +Gag Peptides CD107a Patient #1254 (1 of three patients tested)

21 Conclusions Bortezomib, Sorafenib and Sunitinib reactivate HIV from ART suppressed patient samples Sunitinib reactivates more HIV than Bryostatin and Romodepsin from ART suppressed patient derived cells Sunitinib does not induce transcription or translation of NFƙB/NFAT target proteins Sunitinib may enhance CD8 T cell degranulation in the presence of overlapping gag peptides

22 Future Directions Test for NFƙB Activation in patient CD4 T cells directly Identify the mechanism of Sunitinib, Bortezomid, and Sorafinib dependent HIV reactivation Confirm the enhancement of CD8 T cell degranulation by Sunitinib

23 Acknowledgements Dr. Timothy Henrich Erica Gibson Cassandra Thanh Corinna Schreiner Dr. Louise Hogan Joe Hyatt SCOPE Cohort IAS for the award! You for your attention! Dr. Warner Greene Dr. Jon Chan Dr. Andrea Grammatica Funding Sources: amfar Institute for HIV Cure Research (amfar ) the Delaney AIDS Research Enterprise (DARE; AI and A127966) NIH-NIAID (AI098480; TJH) The SCOPE cohort was also supported by the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763)

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25 Bortezomid does not up-regulate NFkB/NFAT target cell surface (activation) markers Untreated CD3 CD28 IL2 Bortezomid: 4x 8x 16x CD25 CD69

26 HLADR Bortezomid does not up-regulate NFkB/NFAT target cell surface (activation) markers Untreated CD3 CD28 IL2 Bortezomid CD69

27 Even at High Concentrations Sunitinib is Not Toxic Sunitinib (xic50): 0 4x 8x 16x Bryostatin (nm): 10nM Romodepsin (nm): 40nM +Bryostatin 10nM SSC Live Dye

28 Sunitinib increases CD8 T cell degranulation as measured by CD107a expression No Drug Rapamycin 8x Rapamycin 16x Sunitinib 8x Sunitinib 16x CD107a Patient #1763

29 Sunitinib reactivates more 5A8s than Bryostatin and Romodepsin

30 Rapamycin partially inhibits reactivation with low dose Sunitinib in 5A8 cells

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32 Sunitinib Reactivates HIV from 5A8 cells Untreated CD3/CD28 6uL CD3/CD28 12uL CD3/CD28 25uL Sunitinib 4x Sunitinib 8x Sunitinib 16x kb-dsred HIV-GFP

33 5A8s Sunitinib v CD3/CD28

34 Methods HIV + Patient Blood lymphocytes 48 hrs RNA/DNA Extraction From Cells qpcr Cell associated HIV Gag RNA /CCR5 DNA Per 1x10^6 cells 0.5-3x10 6 CD4 T cells/well Sunitinib (4 16 X IC50) Or Bryostatin at 10 or 20 nm w/o Romodepsin 40 nm Cells were pulsed with Romodepsin for 3-4hrs, washed once and then plated w/o Bryostatin