A bis-benzylidine piperidone targeting proteasome ubiquitin receptor RPN13/ADRM1 as a therapy for cancer

Abstract

The bis-benzylidine piperidone RA190 covalently binds to cysteine 88 of ubiquitin receptor RPN13 in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, even those resistant to bortezomib, were sensitive to RA190 via endoplasmic reticulum stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After oral or intraperitoneal dosing of mice, RA190 distributed to plasma and major organs except the brain and inhibited proteasome function in skin and muscle. RA190 administration profoundly reduced growth of MM and ovarian cancer xenografts, and oral RA190 treatment retarded HPV16(+) syngeneic mouse tumor growth, without affecting spontaneous HPV-specific CD8(+) T cell responses, suggesting its therapeutic potential.

Figure 1. RA190 causes a toxic accumulation of polyubiquitinated proteins

(A) RPMI-8226, ANBL6 and their respective in vitro selected bortezomib-resistant cell lines RPMI-8226-V₁₀R and ANBL6-V₁₀R were treated with the indicated compounds for 48 hr and percent cell viability was measured by XTT assay and presented as mean ±SD. (B) The indicated MM cell lines was treated with the indicated compounds for 48 hr and cell viability was measured by XTT assay and presented as mean ±SD. (C) HeLa cells were treated with RA190 (190), RA190ME (190ME) or bortezomib (Bz) for 4 hr (left) or 12 hr (right) at the concentrations indicated and their lysates were probed with anti-K48-linked ubiquitin antibody by immunoblot. An immunoblot of β-tubulin was used to confirm equivalent protein loading. (D) HeLa cells were transiently transfected with either tetraubiquitin-fused firefly luciferase (4UbFL) or FL plasmids. After 48 hr, the transfected cells were treated with various concentrations of the indicated compounds for 4 hr, and luciferase activity was measured. Data is shown as a ratio of 4UbFL to FL and expressed as a fold change ±SD compared to untreated cells. See also Figure S1 and Table S1.

Figure 2. RA190 binds covalently to RPN13

(A) HeLa cell lysate was labeled with RA190B alone or in the presence of competitor RA190 at the indicated concentrations for 1 hr at 4°C. After labeling, equal aliquots were boiled in SDS sample buffer, separated by SDS-PAGE, transferred to a PVDF membrane and probed with streptavidin-peroxidase for the recognition of biotinylated proteins. (B) As for (A) except that the labeling was performed with 500 ng purified 19S proteasome, 10 μM RA190B (190B) and 100 μM RA190 (190). (C) 293TT cells were transfected with plasmid expressing RPN13, RPN10, UCH37 or HHR23B, or luciferase as a control. At 48 hr post-transfection, cell lysates were labeled with RA190B (20 μM), separated by SDS-PAGE, transferred to a PVDF membrane and probed with streptavidin-peroxidase. (D) Lysates of IPTG induced and un-induced bacteria transduced with expression vector for RPN13 or L2 were labeled with RA190B (20 μM), separated by SDS-PAGE, transferred to a PVDF membrane and probed with streptavidin-peroxidase. (E) Competition for labeling of RPN13 expressed in bacterial cell lysate with 200 μM RA190 (190), 20 μM RA190B (190B), or both. (F) The membrane from D was stripped and re-probed with RPN13 antibody. See also Figure S2

Figure 3. RA190 interacts with RPN13 Pru domain

(A, B) Enlarged regions of HSQC spectra for ¹⁵N labeled human RPN13 (A, black) or RPN13 Pru domain (B, black) and after RA190 incubation (A, B, orange). Labels for signals affected by RA190 are italicized and bold. (C) LC-MS experiments for RPN13 Pru domain (top, left) and RA190-exposed RPN13 (top, right), RPN13 Pru domain (bottom, left), and RPN13 Pru C^60,80,121A (bottom, right). The samples used for these experiments are identical to the ones used for the corresponding NMR experiments (A, B and D). (D) HSQC spectra of ¹⁵N labeled RPN13 Pru C^60,80,121A (black) and after RA190 incubation (orange). See also Figure S3.

Figure 4. A surface opposite RPN13 ubiquitin-binding loops is implicated in RA190 binding

(A, B) Normalized peak intensity attenuation (Δ) of RPN13 Pru domain backbone (A) and side chain (B) amide groups upon binding RA190. The dashed line indicates one standard deviation above average. Unassigned, overlapping or proline groups are excluded from this analysis and indicated (*). (C) The lowest energy modeled structure for human RPN13 Pru~RA190 with RPN13 Pru domain colored according to the data in (A and B), such that amino acids most affected by RA190 are highlighted in darkest red (see scale bar for Δ from A). RA190 carbon, nitrogen, oxygen, and chlorine atoms are colored light blue, indigo, red and green, respectively. See also Figure S4.

Figure 5. RA190 upregulates UPR and targets of HPV E6

(A) Immunoblot analysis for ATF-4 and actin in MM.1S cells either untreated (C), or treated with RA190 (190) or bortezomib (Bz) for the indicated times. (B) Immunoblot analysis for ATF-4 in HeLa cells either untreated (C), or treated with 1 μM of RA190 (190) or 1 μM bortezomib (Bz) for 6 hr. (C–E) CHOP-10 (C,D) and XBP1s (E) mRNA levels were assessed by q-RT-PCR and normalized to GAPDH expression after treating HeLa cells with 1 μM RA190 (190) or 1 μM bortezomib (Bz). Results are expressed as fold change over control-treated cells and represent the average and SD of three independent experiments. (F) Immunoblot analysis for Bax protein levels in MM.1S cells treated as for A. (G) Immunoblot analysis for p53 and β-tubulin in the indicated cell line either untreated (C) or treated with 1 μM RA190 (190) or Bortezomib (Bz) for 24 hr (top panel) or for the indicated times (bottom panel). (H) After treatment with 1 μM RA190 (190) or 1 μM bortezomib (Bz) HeLa cell lysates were analyzed by immunoblot for p21, Puma, Bax, Bak and hDLG-1 at the time points indicated. See also Figure S5.

Figure 6. RA190 triggers apoptosis and cell surface presentation of HSP90

(A,B) HeLa cells treated with 1 μM RA190 (190) or botezomib (Bz) for 12 hr (A) or MM.1S cells treated with 0.5 μM RA190 or bortezomib for 6 hr (B) were analyzed by flow cytometry after staining with fluorescein-labeled Annexin-V (C) HeLa cells treated as in (A) were analyzed by flow cytometry after staining for active caspase-3 and the percent of positive cells ±SD was determined. (D) A representative flow cytometry analysis of MM.1S cells treated as in (B) and stained for active caspase-3. (E) Lysates of HeLa cells either untreated (C) or treated with 1 μM RA190 (190) or bortezomib (Bz) after the periods of treatment indicated were immunobloted with PARP-specific antibody. (F) MM.1S cells were untreated (C) or treated with 0.5 μM RA190 or botezomib for 6 hr, then lysed for immunoblot analysis with PARP-specific antibody. (G) HeLa cells were treated with 1 μM RA190, botezomib or cisplatin, and stained at the indicated time points for cell surface HSP90 and analyzed by flow cytometry.

Figure 7. Oral, i.p. and topical administration of RA190 inhibits proteasomal activity in mice

Mice received 4UbFL DNA delivered either i.m. by electroporation (A, C), or intra dermally via gene gun (B, D, E). The transduced mice were treated with vehicle, RA190 or bortezomib (10 mice/group) respectively. After the indicated time points of treatment, bioluminescence was measured by injection of luciferin and imaging with an IVIS 200 (fold change ±SD, * p <0.05, ** p<0.01). RA190 was given i.p. (A, B) or orally (C, D) at 40 mg/kg; bortezomib was given i.p. at 1.5 mg/kg dose (A, B). (E) RA190 was given 4% in Cremophor EL topically. See also Figure S6.

Figure 8. RA190 inhibits tumor growth in vivo

(A) NOG mice carrying NCI-H929-GFP-luc human tumor cells were treated once per day with 20 mg/kg RA190 (i.p.) or vehicle alone (n = 5) on the days indicated. The mice were imaged before and at the end of the treatment for their bioluminescence levels (B) Representative bioluminescence images of mice in (A) before (top panel) and after treatment (bottom panel). (C) Percentage change of bioluminescence ±SD in nude mice bearing ES2-luciferase tumor cells (tumor cells injected i.p.) receiving 10 mg/kg RA190 (i.p.) or vehicle alone (n = 8) every day. Prior to and 7 and 14 days after initiation of treatment the mice were imaged for their luciferase activity. (D) Representative bioluminescence images of mice in (C) before (upper panel) and after treatment (lower panel). (E) Mice bearing palpable TC-1 tumors received oral treatment with 40 mg/kg RA190 or vehicle alone (n = 8 per group) every third day. The mean tumor volumes are plotted ±SD and significant differences indicated (* p<0.05, ** p<0.01). (F) After 2 weeks of treatment, the spleens were harvested. The mean number ±SD of IFNγ⁺ CD8⁺ T cells per 3×10⁵ splenocytes elicited with or without E7 peptide stimulation (E7pep) was determined by flow cytometry and plotted. See also Fig S7 and Table S2 and S3.