Antitumor Activity of Dual BCL-2/BCL-Xl Inhibitor Pelcitoclax (APG-1252) in Natural Killer/T-Cell Lymphoma (NK/TCL)

Abstract Natural killer/T-cell lymphoma (NK/TCL) is one of the most common subtypes (10.4%) of peripheral T-cell lymphoma, which in turn accounts for 10% to 15% of all cases of non-Hodgkin lymphoma. NK/TCL occurs more frequently in Asia and Latin America than other regions, and although associated with Epstein-Barr virus (EBV) infection, NK/TCL has an unclear pathogenesis of genetic and molecular alterations. Asparaginase-based chemotherapy regimens are frequently used, typically with unsatisfactory outcomes. Novel targeted therapies, such as histone deacetylase (HDAC) inhibitors and programmed death-1 antibodies, are reported effective either as single agents or in combination with other agents. Studies have also shown that EBV-positive NK/TCL cell lines are B-cell lymphoma-extra large (BCL-xL) dependent. Other preclinical experiments have shown that BH3-mimetic drugs targeting BCL-xL-induced cell death in NK/TCL cell lines were effective in NK/TCL xenograft models. BCL-xL inhibitors have shown narrow therapeutic windows in clinical trials because of dose-limiting on-target thrombocytopenia. Pelcitoclax (APG-1252) is a novel dual BCL-2/BCL-xL inhibitor under clinical development for solid tumors. As a result of its unique prodrug design, APG-1252 can overcome the undesired platelet toxicity. This study evaluated the potential antitumor effect of APT-1252 in preclinical models of NK/TCL. Cell-based antiproliferation studies showed activity of APG-1252 and its more potent metabolite APG-1252-M1 toward NK/TCL cell lines that overexpressed BCL-xL. Half-maximal inhibitory concentrations (IC 50) for APG-1252 in SNK-1, SNK-6, and SNK-8 (EBV-positive NK/TCL) cell lines were 2.652 ± 2.606 µM, 1.568 ± 1.109 µM, and 0.557 ± 0.383 µM, respectively. Corresponding values for APG-1252-M1 were 0.133 ± 0.056 µM, 0.064 ± 0.014 µM, and 0.020 ± 0.008 µM, respectively. Mechanistic studies demonstrated that APG-1252 and APG-1252-M1 disrupted the complex of BCL-xL/BCL-2-associated X protein (Bax) and BCL-xL/BCL-2 homologous antagonist killer protein (Bak) in SNK-6 cells, thereby liberating these proapoptotic proteins and further activating downstream apoptosis pathways by cleaving poly-ADP ribose polymerase-1 (PARP-1) and caspase-3. In an SNK-6 xenograft model, administration of APG-1252 at 65 mg/kg and 100 mg/kg either twice or once weekly resulted in significant antitumor effects, with tumor growth rate (T/C%) values ranging from 13.7% to 30.7%. Furthermore, the combination of APG-1252 with HDAC inhibitor chidamide or DDGP (dexamethasone, cisplatin, gemcitabine, and pegaspargase) chemotherapy demonstrated synergistic effects. Pharmacokinetic assessment in mice showed that APG-1252 had a long half-life in plasma (127 hours) and tumor tissues (25.2 hours), justifying intermittent dosing schedules used in vivo. Importantly, the transformation of APG-1252 to APG-1252-M1 was 16 times higher in tumor tissues compared to plasma (22% vs. 1.3%) after administration of APG-1252, thereby suggesting that APG-1252 can reduce platelet toxicity caused by APG-1252-M1 in plasma. In conclusion, APG-1252 has promising antitumor effects in NK/TCL, either as a single agent or in combination with an HDAC inhibitor or chemotherapy. These findings provide evidence to further evaluate APG-1252 as a potential treatment for NK/TCL. Disclosures Wang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Liang: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Ming: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Rui: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Tang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. LV: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Ge: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Zhang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Wang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Shang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

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CD55 and CD59 Can Limit the Anti-Tumor Efficacy of Daratumumab in Natural Killer/T-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-115566 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1663-1663 ◽

Cited By ~ 1

Author(s):

Nurulhuda Mustafa ◽

Adina Huey Fang Nee ◽

Jing Yuan Chooi ◽

Sabrina Hui Min Toh ◽

Yan Ting Hee ◽

...

Keyword(s):

T Cell ◽

Natural Killer ◽

Correlation Coefficient ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Resistant Cell ◽

Natural Killer T Cell ◽

Cd38 Expression ◽

Killer T Cell

Abstract Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38. In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and 'Sensitive' tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL. FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either. This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC. To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment. Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. Disclosures Zhou: Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

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Expression of Interleukin-9 in Nasal Natural Killer/T-Cell Lymphoma Cell Lines and Patients

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-05-1426 ◽

2005 ◽

Vol 11 (23) ◽

pp. 8250-8257 ◽

Cited By ~ 51

Author(s):

Toshihiro Nagato ◽

Hiroya Kobayashi ◽

Kan Kishibe ◽

Miki Takahara ◽

Takeshi Ogino ◽

...

Keyword(s):

T Cell ◽

Natural Killer ◽

Cell Lines ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Natural Killer T Cell ◽

Interleukin 9 ◽

Killer T Cell ◽

Natural Killer T

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Oncogenic activation of STAT3 pathway drives PD-L1 expression in natural killer/T cell lymphoma.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.7549 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 7549-7549 ◽

Cited By ~ 3

Author(s):

Soon Thye Lim ◽

Tammy Song ◽

Jing Quan Lim ◽

Yurike Laurensia ◽

Jane Wan Lu Pang ◽

...

Keyword(s):

T Cell ◽

Natural Killer ◽

Cell Lines ◽

Somatic Mutations ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Natural Killer T Cell ◽

Killer T Cell ◽

Stat Pathway ◽

Natural Killer T

7549 Background: Natural killer/T-cell lymphoma (NKTL) is a rare type of non-Hodgkin lymphoma that occurs more frequently in East Asia and Latin America and is associated with Epstein–Barr virus infection. Recent whole-exome sequencing studies in NKTL have reported recurrent somatic mutations in genes associated with JAK-STAT pathway, however the role of aberrant JAK-STAT signaling in tumor immune escape through PD-L1 regulation is unclear. Methods: To determine the prevalence of JAK-STAT pathway alteration in NKTL, we performed targeted sequencing of 188 genes associated with JAK-STAT pathway in 109 NKTL (22 Singapore cases, 79 China cases and 8 cell lines). Single nucleotide variants and micro-indels were called using Freebayes and candidate variants annotated using ANNOVAR. Ba/F3 model system was used to test the transformation capacity of identified variants. Cell lines were evaluated for PD-L1 expression by immunoblotting and flow cytometry. Tissue microarrays were examined for p-STAT3 and PD-L1 expression by immunohistochemistry. Results: We identified a total of 284 non-synonymous somatic mutations candidates in 114 genes, including 243 missense, 10 nonsense, 4 splice-site and 27 indel mutations. Recurrent mutations were most frequently located in STAT3 (25/109 cases, 23%) followed by TP53 (16/109 cases, 16%) and JAK3 (8/109 cases, 7%). A total of 18 STAT3 variants were identified including known hotspot mutations and novel mutations in the SH2, coiled coil and DNA-binding domains. Characterization of novel E616K mutant residing in the SH2 domain showed that E616K conferred IL3 independent growth to Ba/F3 cells, increased STAT3 phosphorylation and PD-L1 expression. Consistent with these findings, PD-L1 was over expressed in cell lines harboring STAT3 mutations. A positive correlation between PD-L1 and p-STAT3 expression was also observed in tumor tissue (R = 0.51, P = 0.02). Conclusions: We characterized a novel activating STAT3 mutant and demonstrated its ability to drive PD-L1 expression, which may promote tumor evasion from the antitumor immune response. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising and novel therapeutic approach for NKTL in the future.

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Expression of CD70 in nasal natural killer/T cell lymphoma cell lines and patients; its role for cell proliferation through binding to soluble CD27

British Journal of Haematology ◽

10.1111/bjh.12136 ◽

2012 ◽

Vol 160 (3) ◽

pp. 331-342 ◽

Cited By ~ 17

Author(s):

Kazumi Yoshino ◽

Kan Kishibe ◽

Toshihiro Nagato ◽

Seigo Ueda ◽

Yuki Komabayashi ◽

...

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Natural Killer ◽

Cell Lines ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Natural Killer T Cell ◽

Killer T Cell ◽

Natural Killer T

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Overexpressed Melk Promotes the Stability of EZH2 through Phosphorylation in Natural Killer/T Cell Lymphoma (NKTL)

Blood ◽

10.1182/blood-2018-99-110758 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2858-2858

Author(s):

Boheng Li ◽

Dennis Kappei ◽

Junli Yan ◽

Pieter Eichhorn ◽

Siok Bian Ng ◽

...

Keyword(s):

T Cell ◽

Natural Killer ◽

Research Funding ◽

Cell Lymphoma ◽

Mrna Level ◽

T Cell Lymphoma ◽

Natural Killer T Cell ◽

The Impact ◽

Killer T Cell ◽

Natural Killer T

Abstract EZH2 oncogene is extensively involved in pathophysiology of different cancer contexts including natural killer/T cell lymphoma (NKTL). Only a few studies have dealt with regulations of EZH2 stability in specific context, and it remains un-targetable in NKTL. EZH2 is over-expressed in NKTL and the mechanism is unclear. In this study, we examined EZH2 protein turnover mechanisms in the NKTL context. The serine/threonine kinase Melk is one of the overexpressed genes in NKTL patient samples and cell lines, and the interaction between Melk and EZH2 was established by co-immunoprecipitation. Inhibition of Melk using inhibitor or siRNA both resulted in a decrease of EZH2 protein levels in NKTL cells, whereas there was no change in the mRNA level of EZH2, suggesting that Melk regulated EZH2 at the protein level. Next, we observed a change of EZH2 ubiquitination upon manipulation of Melk expression. Next, in order to confirm that Melk truly affect EZH2 ubiquitination and to identify its (de)ubiquitination site, we used a SILAC-based mass spectrometry (MS) approach. We overexpressed all-lysine-mutated ubiquitin plus EZH2 with or without Melk overexpression in 293T cells, and pulled down EZH2 for MS analysis. The MS data found a decrease of K48-linked ubiquitin peptide upon Melk overexpression, which corresponded to the impact of Melk on EZH2 protein stability, as well as two possible sites of EZH2 (de)ubiquitination. One of these two sites were confirmed in later experiments as a critical site (K222). As Melk is a kinase, it is possible that the regulation of EZH2 ubiquitination is phosphorylation-based. A re-analysis of the EZH2 post-translational modification from the MS data identified S220-phosphorylated EZH2 upon Melk overexpression. And by comparing the forward and reverse H/L ratio of S220-phospho-EZH2 and K222-ubiquitinated-EZH2 peptide, we found the phosphorylation should be an earlier event before (de)ubiquitination. Correspondingly, the enzymatic-dead mutant of Melk could rescue the deubiquitination effect of Melk wildtype on EZH2. The Melk-mediated deubiquitination of EZH2 also mediates Velcade resistance in NKTL. EZH2 overexpression led to increased resistance to Velcade treatment, which could be rescued by EZH2 S220 phospho-dead mutant transfection, and promoted by EZH2 K222 ubi-dead mutant transfection. Conversely, Melk knock-down sensitized the NK lymphoma cells to Velcade treatment. Collectively, this study uncovered a role of Melk in mediating EZH2 ubiquitination through phosphorylation and thus regulating resistance to Velcade in NKTL context. Disclosures Chng: Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

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