LNS-8801 as a therapeutic agent for aggressive lymphomas: ROS-induced cytotoxicity and synergy with existing therapies Open Access

TO THE EDITOR:

Aggressive lymphomas constitute a heterogeneous group of hematologic malignancies arising from transformed lymphocytes of various developmental stages and are frequently associated with significant unmet clinical needs, particularly in relapsed and refractory cases.^1,2 Both diffuse large B-cell lymphoma (DLBCL) and the CD30⁺ peripheral T-cell lymphoma anaplastic large cell lymphoma (ALCL) show a higher incidence in men, indicating a role for sex hormones in the development of these diseases.^3,4 Especially estrogen, which can be recognized by the nuclear estrogen receptors alpha and beta or by membrane estrogen receptors such as G protein–coupled estrogen receptor 1 (GPER), has gained increasing attention in lymphoma research. For example, activation of estrogen receptor beta is associated with impaired proliferation, vascularization, and dissemination of mantle cell and Burkitt lymphoma.^4,5 Given that GPER expression is not restricted to reproductive organs but detectable in many tissues and cell types and its activation has been reported to exert tumor suppressive effects in solid tumors such as melanoma or pancreatic ductal adenocarcinoma,^6,7 we investigated the antilymphoma potential of the selective GPER agonist LNS-8801.⁸ 

To test the efficacy of LNS-8801 in hematologic malignancies in general, we initially evaluated the antitumor effects of LNS-8801 as monotherapy in 4 hematologic cancer entities: ALCL, DLBCL, multiple myeloma, and acute lymphoblastic leukemia. LNS-8801 demonstrated potent antitumor activity in 3 of the 4 models investigated, with the most pronounced effects seen in the lymphoma entities ALCL (median 50% inhibitory concentration [IC₅₀] = 245 nM) and DLBCL (median IC₅₀ = 411 nM) (Figure 1A; all methods are described in the supplemental Methods). Notably, the sensitivity to LNS-8801 was not restricted to a specific subtype within ALCL, given that reduced viability was observed in 4 ALK-positive and 5 ALK-negative ALCL cell lines (supplemental Figure 1A; supplemental Table 1). Similarly, both activated B-cell–like and germinal center B-cell–like DLBCL subtypes responded to LNS-8801 treatment in the nanomolar range (supplemental Figure 1B; supplemental Table 1). Taken together, these data indicate that LNS-8801 has broad efficacy against ALCL and DLBCL regardless of their molecular subtype classification.

To investigate whether GPER expression correlates with the cytotoxic effect of LNS-8801 in ALCL and DLBCL cell lines, we analyzed GPER expression by quantitative polymerase chain reaction. Interestingly, we were not able to detect any significant correlations between GPER expression levels and the IC₅₀ values of LNS-8801 (Figure 1B; supplemental Figure 1C). To validate whether the cytotoxic effect of LNS-8801 is mediated by GPER activation in our lymphoma models, we cotreated 2 ALCL and DLBCL cell lines, respectively, with the specific GPER antagonist G15.^9,10 However, the additional administration of the GPER antagonist was unable to attenuate the cytotoxicity induced by LNS-8801 (Figure 1C), suggesting a GPER-independent antilymphoma effect of LNS-8801. Accordingly, GPER knockout Mac-2A exhibited a similar susceptibility to LNS-8801 as the parental line (Figure 1D). In conclusion, these data demonstrate that LNS-8801 has a broad antilymphoma effect, but its cytotoxicity is independent of GPER in ALCL and DLBCL.

To shed light on the molecular mechanisms underlying LNS-8801–mediated cytotoxicity, we investigated whether the lymphoma cells undergo apoptosis upon treatment. Indeed, an increased frequency of annexin V–positive/propidium iodide–negative cells, as well as caspase activation, was detectable in most LNS-8801–treated ALCL and DLBCL cell lines (supplemental Figure 2A-B). Accordingly, the addition of Q-VD-OPH, a pan-caspase inhibitor, significantly reduced both cleaved caspase levels and LNS-8801–induced cell death in the sensitive ALCL and DLBCL models, further substantiating a functional role of apoptosis induction for LNS-8801–mediated cytotoxicity in the lymphoma cells (supplemental Figure 2C-D). Given that it has been reported that LNS-8801 treatment can provoke the generation of reactive oxygen species (ROS) in acute myeloid leukemia cell lines, we quantified ROS levels by flow cytometry.¹¹ After LNS-8801 treatment, we detected increased ROS levels in sensitive ALCL and DLBCL cell lines (Figure 2A). To investigate the importance of ROS generation for LNS-8801-induced toxicity, we added the antioxidant α-tocopherol to the treatment. α-Tocopherol was not only able to reduce intracellular ROS levels but also almost completely protected ALCL cell lines and at least partially rescued DLBCL cell lines from the cytotoxic effect of LNS-8801 (Figure 2B-C; supplemental Figure 3A-B). Given that the mitochondria are a major source of ROS in cells, we analyzed the fitness of these organelles by measuring the mitochondrial membrane potential. We observed mitochondrial depolarization in the LNS-8801-treated sensitive lymphoma cells, which could be reversed by the addition of α-tocopherol (Figure 2D; supplemental Figure 3C). These data suggest that oxidative stress induction by LNS-8801 leads to mitochondrial damage, thereby promoting apoptosis induction.

To evaluate whether the antilymphoma activity of LNS-8801 could improve current treatment regimens for ALK-negative and -positive ALCL, we combined it with the anti-CD30 antibody-drug conjugate (ADC) brentuximab vedotin^12,13 or with the ALK inhibitor crizotinib,^14,15 respectively. In both cases, the combinatorial treatments resulted in synergistic killing of the ALCL cells, suggesting that LNS-8801 is indeed able to improve the efficacy of these drugs (Figure 2E; supplemental Figures 4A and 5). Based on the finding that LNS-8801 synergizes with brentuximab vedotin in ALCL, we tested its influence on the efficacy of the CD79b-targeting ADC polatuzumab vedotin, which is currently used for the treatment of patients with untreated or relapsed/refractory DLBCL.^16,17 Similar to our previous results in ALCL, LNS-8801 enhanced the efficacy of the ADC in the tested DLBCL cell lines (Figure 2F; supplemental Figure 4B-C). Furthermore, the synergy of the combinatorial treatment was detectable regardless of the cell lines’ classification as germinal center B-cell–like or activated B-cell–like DLBCL.

Given that microtubule-targeting agents, such as monomethyl auristatin E, which is part of the vedotin moiety in both ADCs, have been associated with ROS formation, we analyzed ROS levels after monotherapy with the ADC or in combination with LNS-8801.¹⁸ Although low concentrations of the single agents provoked only limited ROS production, the combinatorial treatment resulted in elevated ROS levels (supplemental Figure 6A). Given that similar effects were observed in the ALK-positive ALCL cell line JB6 after treatment with LNS-8801 and crizotinib combinations, we propose that the molecular basis by which LNS-8801 enhances the efficacy of various antilymphoma treatments lies in its ability to boost ROS generation (supplemental Figure 6B).^19,20 

Interestingly, LNS-8801 exhibits broad anticancer activity in pancreatic ductal adenocarcinoma, malignant melanoma, and leukemia.^6,7 However, there is an ongoing debate regarding its mode of action. In various solid tumors, the tumor-suppressing effects of LNS-8801 are described as GPER dependent, whereas in acute myeloid leukemia it seems GPER independent.^6-8,11 Building on these findings and our data, we propose that the effect of LNS-8801 in hematologic malignancies is not primarily based on the activation of GPER, but rather on its ability to elevate intracellular ROS levels. How LNS-8801 boosts ROS generation on the molecular level is not yet clear, but because we could block mitochondrial depolarization by the addition of an antioxidant, we do not expect that it disrupts the electron transfer chain or the mitochondrial integrity.

In conclusion, LNS-8801 demonstrates strong in vitro antitumor efficacy in the nanomolar range against different aggressive lymphomas such as ALCL and DLBCL. Surprisingly, this activity is independent of GPER, but relies, at least partially, on ROS production. Combination of LNS-8801 with approved clinical agents such as ADCs or crizotinib potentiates ROS production and cytotoxicity in ALCL and DLBCL models. Our findings underscore the potential of LNS-8801 to improve the current treatment of aggressive lymphomas and provide a rationale for further testing of its effectiveness in hematologic malignancies.

Acknowledgments: The authors thank Marshall Kadin and Jürgen Eberle for providing the Mac-1 cell line and Linnaeus Therapeutics for their kind donation of LNS-8801 for use in the study.

Contribution: Y.L. designed research, performed experiments, analyzed data, and wrote the manuscript; C.K. analyzed data and wrote the manuscript; D.S. and L.J. performed experiments; M.G. analyzed data; S.H. and G.L. designed research, analyzed data, and wrote the manuscript; and all authors read and approved the final manuscript.

Conflict-of-interest disclosure: G.L. received research grants not related to this manuscript from Agios, Aquinox, AstraZeneca, Bayer, Celgene, Gilead, Janssen, MorphoSys, Novartis, F. Hoffmann-La Roche Ltd, and Verastem. G.L. received honoraria from ADC Therapeutics, AbbVie, Amgen, AstraZeneca, Bayer, BeiGene, Bristol Myers Squibb, Celgene, Constellation, Genase, Genmab, Gilead, Hexal/Sandoz, Inmagene, Incyte, Janssen, Karyopharm, Lilly, Miltenyi Biotec, MorphoSys, Merck Sharp & Dohme, NanoString, Novartis, Pentixapharm, Pierre Fabre, F. Hoffmann-La Roche Ltd, and Sobi. The remaining authors declare no competing financial interests.

Correspondence: Stephan Hailfinger, Department of Hematology, Oncology and Pneumology, University Hospital Muenster, Domagkstr. 3, 48149 Muenster, Germany; email: stephan.hailfinger@ukmuenster.de.