Abstract Therapeutic strategies based on exacerbated stress signaling may represent novel and effective treatment paradigms in oncology. Here, we disclose for the first time the endoplasmic reticulum (ER) stress modulator (ERSM) M3913 that was discovered and developed following a phenotypic screening campaign. Mechanistically, M3913 engages an ER transmembrane protein not yet implicated in cancer biology to induce a Ca2+ shift from the ER towards the cytoplasm, resulting in an unfolded protein response and subsequent antitumor activity in sensitive preclinical models. We detected concurrent elevation of bona fide ER stress markers by several methods including qRT-PCR, Western Blotting analysis, and unbiased transcriptome analysis. Genetic removal of M3913’s putative ER transmembrane target abrogated the M3913-mediated, but not the Thapsigargin-mediated, ER stress response. PK/PD studies confirmed a dose- and time-dependent upregulation of ER stress markers in preclinical cancer models in vivo. As a monotherapy, M3913 induced full and partial tumor regression in preclinical models of multiple myeloma, non-small-cell lung cancer, triple-negative breast cancer, and other cancers. An in vitro combination screen indicated combination potential of M3913 with standard-of-care agents and novel candidate drugs. IND-enabling toxicology and additional mechanism-of-action studies in rat and minipig uncovered specific target organs for M3913 in line with expression of the target in human tissues. This presentation details mechanistic profiling data from M3913, reveals its putative ER-resident target and discusses forward translation studies that support M3913 as a novel therapeutic option for hard-to-treat subtypes of a variety of cancers. Citation Format: Frank Czauderna, Richard Schneider, Shivapriya Ramaswamy, Susanne Brandstetter, Elise Drouin, Olga Bogatyrova, Johanna Mazur, Qing Sun, Catherine Jorand-Lebrun, Eva Sherbetjian, Jonny Nachtigall, Carolyn Wing, Christian Hildebrand, Peter Ellinghaus, Isabelle Lemoine, Mac Johnson, Russell Hoover, Michael Clark, Ralph Lindemann. M3913 induces the maladaptive unfolded protein response through a novel mechanism resulting in strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND09.
The majority of breast cancers metastasizing to bone secrete parathyroid hormone-related protein (PTHrP). PTHrP induces local osteolysis that leads to activation of bone matrix-borne transforming growth factor β (TGFβ). In turn, TGFβ stimulates PTHrP expression and, thereby, accelerates bone destruction. We studied the mechanism by which TGFβ activates PTHrP in invasive MDA-MB-231 breast cancer cells. We demonstrate that TGFβ1 up-regulates specifically the level of PTHrP P3 promoter-derived RNA in an actinomycin D-sensitive fashion. Transient transfection studies revealed that TGFβ1 and its effector Smad3 are able to activate the P3 promoter. This effect depended upon an AGAC box and a previously described Ets binding site. Addition of Ets1 greatly enhanced the Smad3/TGFβ-mediated activation. Ets2 had also some effect, whereas other Ets proteins, Elf-1, Ese-1, and Erf-1, failed to cooperate with Smad3. In comparison, Ets1 did not increase Smad3/TGFβ-induced stimulation of the TGFβ-responsive plasminogen activator inhibitor 1 (PAI-1) promoter. Smad3 and Smad4 were able to specifically interact with the PTHrP P3-AGAC box and to bind to the P3 promoter together with Ets1. Inhibition of endogenous Ets1 expression by calphostin C abrogated TGFβ-induced up-regulation of the P3 transcript, whereas it did not affect the TGFβ effect on PAI expression. In TGFβ receptor II- and Ets1-deficient, noninvasive MCF-7 breast cancer cells, TGFβ1 neither influenced endogenous PTHrP expression nor stimulated the PTHrP P3 promoter. These data suggest that TGFβ activates PTHrP expression by specifically up-regulating transcription from the PTHrP P3 promoter through a novel Smad3/Ets1 synergism.
<p>PDF file - 209K, Everolimus is associated with changes to the immunophenotype. SF8: Continued daily dosing with everolimus results in cell cycle arrest</p>
Transcription factor Ets1 is expressed in invasive breast cancer cells. In T-cells, the splicing variant DeltaVII-Ets1 is naturally produced along with full length Ets1 (fl-Ets1). Though its function is unknown, the lack of important inhibitory domains predicts a regulatory role of DeltaVII-Ets1 in fl-Ets1-controlled transcription. Examining the expression status of DeltaVII-Ets1 in invasive Ets1-producing MDA-MB-231 breast cancer cells, we found that the DeltaVII-Ets1 protein could only be detected when nuclear proteins from these cells were fractionated by ionic exchange chromatography. When overexpressed, DeltaVII-Ets1 was found to be partially degraded in breast cancer cells, but not in Jurkat T-cells or SK-Mel melanoma cells. In contrast, no proteolytic products resulted from ectopic expression of fl-Ets1 suggesting that breast cancer cells are able to specifically cleave DeltaVII-Ets1. Overexpression of DeltaVII-Ets1 reduced survival of MDA-MB-231 cells, but not of MCF-7 cells. A mutant version of DeltaVII-Ets1, lacking first 129 N-terminal amino acids, had no effect. These data suggest that Ets1-producing invasive breast cancer cells specifically downregulate DeltaVII-Ets1, as it may be able to adversely affect the survival of these cells.
Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival including inhibition of tumor cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis and modulation of immune responses and show promising activity against hematological malignancies in clinical trials. Using the Eµ-myc model of B-cell lymphoma and the HDACi LBH589 and LAQ824 we demonstrated a direct correlation between induction of tumor cell death in vivo and therapeutic efficacy. Neither HDACi required p53 activity or a functional death receptor pathway, but mediated lymphoma cell death via the intrinsic apoptotic pathway as demonstrated by decreased apoptosis and therapeutic activity of LBH589 and LAQ825 against Eµ-myc/Bcl-2 and Eµ-myc/Bcl-XL cells. Interestingly, both LBH589 and LAQ824 effectively killed Eµ-myc/caspase-9-/- and Eµ-myc/Apaf-1-/- lymphomas, which lack a functional apoptosome and thus have a defective apoptotic program downstream of the mitochondria. These cells did not display classic morphological or biological features of apoptosis following treatment with LBH589 and LAQ824 however their clonogenic capacity was significantly reduced and interestingly electron microscopy analysis indicated that these HDACi-treated cells underwent autophagy. Importantly, both Eµ-myc/caspase-9-/- and Eµ-myc/Apaf-1-/- responded to LBH589 and LAQ824 in vivo indicating that in the absence of an effective apoptotic program downstream of mitochondrial membrane perturbation, tumor cells can undergo autophagy and this is sufficient to mediate therapeutic efficacy. Our studies provide important information regarding the mechanisms of action of LBH589 and LAQ824 that may have broader implications regarding future stratification of patients receiving therapy with these agents and the use of these compounds in combination with other anti-cancer agents.
Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.
<p>PDF file - 209K, Everolimus is associated with changes to the immunophenotype. SF8: Continued daily dosing with everolimus results in cell cycle arrest</p>
<p>PDF file - 1.1MB, Quantitation of SA-B-gal and immunostaining. SF10: Markers of senescence. SF11: Overall survival of mice transplanted with a p53 mutant lymphoma</p>
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
