Remi Delansorne

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC50 in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.[Mol Cancer Ther 2009;8(8):2286–95]

Inecalcitol, an analog of 1,25D₃, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system

Epidemiological data suggest an important role of vitamin D signaling in cancer development and progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D₃ (1,25D₃) has broad spectrum antitumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D₃, inecalcitol [19-nor-14-epi-23-yne-1,25-(OH)₂D₃; TX522], was developed to have superagonistic antitumor activities but low hypercalcemia potential. We examined the antitumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. In vitro, compared with 1,25D₃, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose-dependent manner with an IC₅₀ value 30 times lower than that of 1,25D₃. Both inecalcitol and 1,25D₃ induced a comparable level of G₀/G₁ cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D₃. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared with 1,25D₃. In vivo, inecalcitol inhibits SCC tumor growth in a dose-dependent fashion. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. While in vitro inecalcitol demonstrates apparent enhanced VDR binding and antiproliferative effects compared to 1,25D₃, in vivo these advantages disappear; at doses of inecalcitol that have equivalent antitumor effects, similar hypercalcemia is seen. This may be explained by the pharmacokinetics of 1,25D₃ vs. inecalcitol and attributed to the much shorter serum half-life of inecalcitol.We show that inecalcitol has potent antitumor activity in the SCC model system, and this is associated with a strong induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment.

USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status

The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised.

Abstract 1890: Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6

Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38 - cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38 + cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38 - HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38 + cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38 - cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38 - AML cell lines and the density of CD38 increased at the surface of CD38 + AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab. Citation Format: Enguerran Mouly, Cecile Planquette, Emilie Rousseau, Remi Delansorne. Inecalcitol respectively induces or increases CD38 expression at the surface of CD38 - or CD38 + AML cell lines representative of all 9 FAB subtypes except M6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1890.

Abstract 2633: Vitamin D3 analog inecalcitol synergizes with tyrosine kinase inhibitors (TKI) and selectively inhibit the growth of chronic myeloid leukemia (CML) progenitors: Development of a clinically applicable leukemic stem cell targeting strategy

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history of chronic myeloid leukemia (CML) and prolonged survival. However, in vitro and in vivo data suggest strongly that the eradication of the most primitive CML stem cells will not be possible by the use of TKI therapies alone. The mechanisms of this inefficiency might involve cell autonomous (activation of alternate signaling, reduced BCR-ABL expression) or non-cell autonomous (niche-related) pathways. It would therefore be of major interest to determine if compounds targeting CML progenitors and stem cells can be used in combination with TKI. Few targeted therapies have been so far shown to be clinicaly acceptable. We have used for this purpose Inecalcitol (19, nor 14 epi 23-yne-1,25 (OH)2D3) (ICC),a vitamin D3 analog, which has been shown to exert antiproliferative effects in several types of cancer cell lines. CD34+ cells isolated from CML patients at diagnosis (n = 15) were tested in clonogenic assays (500 CD34+ cells / dish in triplicate). Interestingly, ICC alone is highly efficient to inhibit the clonogenic growth in the majority of the CML patients at diagnosis (10/ 15 patients). The combination of ICC with either Imatinib Mesylate (IM), Dasatinib (DA) or Nilotinib (NIL) in clonogenic assays showed a synergistic effect for the inhibition of CFC growth (10 - 25% CFC survival) using IM (n = 15 patients). In the same conditions, we have not observed any significant inhibitory effect of IM and ICC combination in cord-blood derived progenitors (n = 3). To determine the effects of these combinations in the most primitive stem cells, we have performed long-term cultures initiating cell (LTC-IC) assays using purified CD34+ cells (4.104 cells / dish) from CML patients with half medium changes each week for 5 weeks. The combination was tested in 6 (ICC+ IM) and 4 (ICC + DA / NIL) CML samples. As a control, CD34+ cells from cord blood were used. In CML samples tested with the combination of either IM / ICC (n = 6) or DA/ NIL and ICC (n = 4), CML LTC-IC derived progenitors were highly inhibited (ICC and IM) or undetectable (ICC and DA or NIL). Short-term cultures of CML CD34+ cells in the presence of 5 growth factors with or without ICC showed that ICC induced the expression of myeloid markers and highly favored the appearance of double positive CD14/CD15 cells. Experiments are underway to determine if ICC interferes with the expression of the components of SHH pathway (Smo, Ptched, Gli). as well as the gene expression profiling of CML cells treated with ICC. Thus, these results establish that ICC, a clinically used derivative of vitamin D3 has a clear activity in CML progenitors by itself and a major synergistic effect with TKI. A clinical phase 2 trial aiming to confirm the synergistic effect of ICC and TKI is ongoing in CML patients treated with imatinib. Citation Format: Ali G. Turhan, Hyacinthe Johnson Ansah, Patricia Hugues, Camille Debord, Remi Delansorne, Agnes Guerci-Bresler, Jean Francois Dufour Lamartinie, Annelise Bennaceur-Griscelli. Vitamin D3 analog inecalcitol synergizes with tyrosine kinase inhibitors (TKI) and selectively inhibit the growth of chronic myeloid leukemia (CML) progenitors: Development of a clinically applicable leukemic stem cell targeting strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2633. doi:10.1158/1538-7445.AM2015-2633

Abstract 3434: Inecalcitol, an analogue of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system.

Epidemiological data suggest an important role of vitamin D signaling in cancer progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D3 (1,25D3) has broad spectrum anti-tumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D3, inecalcitol (19-nor-14-epi-23-yne-1,25-(OH)2D3; TX522), was developed to have superagonistic anti-tumor activities but low hypercalcemia potential. We examined the anti-tumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. Compared to 1,25D3, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose dependent manner with an IC50 value 30 times lower than that of 1,25D3. Both inecalcitol and 1,25D3 induced a comparable level of G0/G1 cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D3. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared to 1,25D3. In vivo, inecalcitol inhibits SCC tumor growth at 80 μg dose without hypercalcemia. Inecalcitol (320 μg) or 1,25D3 (0.312 μg) markedly inhibited tumor growth. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. We show that inecalcitol has potent anti-tumor activity in the SCC model system and this is associated with a stronger induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment. Citation Format: Yingyu Ma, Wei-Dong Yu, Alejandro A. Hidalgo, Wei Luo, Remi Delansorne, Candace S. Johnson, Donald L. Trump. Inecalcitol, an analogue of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3434. doi:10.1158/1538-7445.AM2013-3434

Abstract 583: Inecalcitol, a 14-epi-analogue of 1,25D 3 , induces growth inhibition through apoptosis and cell cycle arrest in a squamous cell carcinoma model system

Epidemiological and experimental studies have demonstrated that 1,25D 3 , the active vitamin D metabolite, has broad spectrum anti-tumor activity. However, hypercalcemia may limit its clinical application when administrated inappropriately. An orally available 14-epi-analogue of 1,25D 3 , inecalcitol (19-nor-14-epi-23-yne-1,25-(OH) 2 D 3 , TX522), has shown good clinical tolerance at high and frequent exposure without side effects and hypercalcemia. Therefore, we examined the antitumor activity of inecalcitol in a squamous cell carcinoma (SCC) model system. Inecalcitol at 10 or 100 nM readily induced vitamin D receptor expression in SCC cells. Inecalcitol suppressed SCC cell proliferation in a dose dependent manner as shown by MTT assays. SCC cells underwent G0/G1 cell cycle arrest following inecalcitol treatment, which was accompanied by the induction of p27 protein as shown by immunoblot analysis. Inecalcitol markedly induced apoptosis in SCC cells as examined by annexin V/7AAD staining and DNA fragmentation ELISA assays. The cleavages of caspases 8, 9, and 3 and PARP were observed following inecalcitol treatment in SCC cells. In addition, migration assay revealed that inecalcitol inhibited the motility of SCC cells. Inecalcitol also suppressed the invasion of SCC cells as assessed by Matrigel-based invasion assay. To have a better understanding of the effects of inecalcitol, 1,25D 3 -resistant variant of SCC (SCC-DR) were employed. The above findings were not observed in SCC-DR cells, indicating the critical role of inecalcitol. Our studies suggest that inecalcitol strongly suppress SCC growth through the promotion of apoptosis and cell cycle arrest. Inecalcitol also inhibits SCC migration and invasion. Thus, inecalcitol may be a promising 1,25D 3 analogue for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2011-583

Abstract 2642: Identification and characterization of selective inhibitors of ubiquitin specific protease 7

Regulated protein turnover is primarily controlled by the ubiquitin-proteasome system. The only marketed drug related to the ubiquitin-proteasome system, bortezomib, is acting as a proteasome inhibitor and has been approved for the treatment of some hematological cancers. Targeting the upstream ubiquitin conjugation/deconjugation system carries out promises of therapeutics with increased specificity and selectivity. Ubiquitin-specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a drugable target class due to their thiol-protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome-wide RNAi screen of the catalytically active human USPs in cancer-relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP as promising cancer target. Fluorescence-based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High-throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP7 inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP7-specific series. These will help further validate this novel class of molecular targets and may provide a structural basis for the development of new anticancer drugs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2642.

Abstract B193: Identification of selective inhibitors of ubiquitin specific proteases

Regulated protein turnover is primarily controlled by the ubiquitin‐proteasome system. The therapeutic efficacy of the proteasome inhibitor Bortezomib establishes this system as a valid anticancer therapeutic field. A promising alternative to targeting the proteasome itself lies upstream, at the level of the ubiquitin conjugation/deconjugation step to generate more specific anticancer agents. Ubiquitin‐specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a druggable target class due to their thiol‐protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome‐wide RNAi screen of the catalytically active human USPs in cancer‐relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP and USP8/UBPY as promising cancer targets. Fluorescence‐based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High‐throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP8‐ and USP7‐specific series. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B193.