Search

Supplementary Data including Table S1-Table S5 and Figure S1 - Figure S9

PDF file - 213K

PDF file - 1.2MB

The following information of relevance to the manuscript is included: The source and authentication of all cell lines is included in Supplementary Table S1 and is of importance for validation of the results described in the manuscript. Data showing the mutual exclusivity and co-occurrence of IGF2 overexpression with other common molecular alterations found in CRC is included in Supplementary Tables S2 and S3, respectively. This information is of importance for understanding the different oncogenic pathways that are commonly present in IGF2-overexpressing CRC tumors. The sensitivity of cell lines to two structurally divergent IGF1R/INSR inhibitors is shown in Supplementary Table S4. This is of importance to validate that the BI 885578 sensitivity of the cell lines is related to IGF1R/INSR inhibition and not the inhibition of potential BI 885578 off-targets. The in vitro sensitivity data for non-CRC cell lines is shown in Supplementary Table S5 which corresponds to the data presented in Figure 6. This is of importance for the reader to have access to the GI50 data for each cell lines tested. The expression of IGF2 on a separate data set from TCGA is shown in Supplementary Figure S1 and is of importance to validate the frequency of IGF2 overexpressing colon tumors. The expression of total IGF1R and total Akt protein by CRC cell lines is shown in Supplementary Figure S2 and this is of importance as it demonstrates that the absence of phospho-IGF1R or phospho-Akt signals in some cell lines is not due to low expression of the corresponding total proteins. The effect of BI 885578 on phospho-IGF1R and phospho-Akt levels in vivo is shown in Supplementary Figure S3 to demonstrated that dosing of the compound led to engagement of the IGF pathway in vivo. The tumor growth kinetics are shown in Supplementary Figure S4 order to demonstrate the time-course of response of the different tumor models to BI 885578, anti-VEGF-A and the combination of these compounds. The change in mouse body weight data is shown in Supplementary Figure S5 to confirm that BI 885578, anti-VEGF and the combination of these compounds were tolerated. The efficacy of BI 885578 in combination with diverse agents used in CRC is shown in Supplementary Figure S6 and has relevance for identification of the most promising combinations that could potentially be applied in future clinical studies with BI 885578. The change in mouse body weight data is shown in Supplementary Figure S7 to confirm that the compounds used in this study were tolerated. The effect of BI 885578 on HIF1-alpha and VEGF-A expression is shown in Supplementary Figures S8 and S9, respectively. This has relevance for understanding the role of IGF2 in promoting the expression of these pro-angiogenic proteins. The expression of IGF1R protein by non-CRC cell lines is shown in Supplementary Figure S10 and this is of importance as it demonstrates that the absence of phospho-IGF1R signals in some cell lines is not due to low expression of total IGF1R.

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell–mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti–PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell–mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.

Supplementary Fig. S1 from PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer

Supplemental Materials & Methods and the references cited within.

Legends of supplemental figures S1-S5

Supplementary Table S1 from PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer

Overview of human testis biopsy samples, which were analyzed for RHAMM parameters (mRNA expression, protein localization, locus variants, allele frequencies) as well as for expression of CFIm25 and of 12 TGCT susceptibility genes.

Its centrosome-targeting domain is indispensable for RHAMM spindle localization and it contributes to maintenance of spindle integrity in vitro.

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition–related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status–dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser10 is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers. [Mol Cancer Ther 2007;6(12):3158–68]

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM—a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self‐renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382–95. ©2016 AACR.

Supplementary Figures 1-14, Tables 1-4, Methods, References from Targeting the Mitotic Checkpoint for Cancer Therapy with NMS-P715, an Inhibitor of MPS1 Kinase

Replicative senescence limits the number of times primary cells can divide and is therefore regarded as a potential checkpoint for cancer progression. The majority of studies examining changes of gene expression upon senescence have been made with stationary senescent cells. We wanted to study the transition from normal growth to senescence in detail and identify early regulators of senescence by analyzing early changes in global gene expression, using Affymetrix microarrays. For this purpose, we used a murine epithelial senescence model, where senescence is abrogated by SV40 large T antigen and can be induced by using a temperature-sensitive form of SV40 large T antigen (SV40ts58). Comparisons were made to wild-type SV40 large T antigen-expressing cells and to cells expressing SV40ts58 large T antigen grown to confluence. After removal of genes that are similarly regulated in wild-type and temperature-sensitive SV40 large T antigen-expressing cells, 60% of the remaining genes were shared between cells arrested by inactivation of SV40 T antigen and by confluence. We identified 125 up-regulated and 39 down-regulated candidate genes/expressed sequence tags that are regulated upon SV40 T antigen inactivation and not during heat shock or confluence and classified these based on their kinetic profiles. Our study identified genes that fall into different functional clusters, such as transforming growth factor-β-related genes and transcription factors, and included genes not identified previously as senescence associated. The genes are candidates as early regulators of the senescence checkpoint and may be potential molecular targets for novel anticancer drugs.

11.5 MB Table in Microsoft Excel

Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine. [Cancer Res 2007;67(17):7987–90]

Supplementary Table 1 from Crystal Structure of the T315I Abl Mutant in Complex with the Aurora Kinases Inhibitor PHA-739358

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell–mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti–PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell–mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.

xCT is a cystine/glutamate antiporter that exports intracellular glutamate for extracellular cystine. In the intracellular space, cystine is converted into cysteine which is subsequently used for glutathione (GSH) synthesis (a major antioxidant species). ARID1A is part of the SWI-SNF remodeling complex that binds on the xCT promoter and as such controls its gene expression. ARID1A deficiency which is highly prevalent in many cancers results in downregulation of xCT, impaired GSH biosynthesis and subsequent ROS induction, raising potential rationale to target xCT in ARID1A deficient cancers. By using a selective nanomolar range xCT inhibitor, we first showed that cell lines deficient for ARID1A were significantly more sensitive to the inhibition of xCT. Effects on proliferation could be reversed by N-acetylcysteine supplementation in the culture medium. In vivo, the xCT inhibitor confirmed its drug-like properties showing significant target engagement in an ARID1A deficient ovarian cell line xenograft model together with good PK and tolerability profiles. Altogether these studies have provided evidence that a specific drug-like inhibitor of xCT can be developed. This proprietary compound was used to confirm that in vitro the deficiency of ARID1A in ovarian cancer predicts sensitivity to xCT inhibition. Further in vivo pharmacological studies are required to confirm a specific molecular context predicting sensitivity towards xCT inhibition. Citation Format: Christophe Henry, Philippe Péron, Anne-Marie Blanchet, Arielle Genevois-Borella, Marc Trellu, Dorothée Bourges, Philippe Lienard, Alexandra Basset, Erwan Jouannot, Christophe Philippo, Laurent Debussche, Jürgen Moll. Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 130.

Summary Misoriented division of neuroprogenitors, by loss-of-function studies of centrosome or spindle components, has been linked to the developmental brain defects microcephaly and lissencephaly. As these approaches also affect centrosome biogenesis, spindle assembly, or cell-cycle progression, the resulting pathologies cannot be attributed solely to spindle misorientation. To address this issue, we employed a truncation of the spindle-orienting protein RHAMM. This truncation of the RHAMM centrosome-targeting domain does not have an impact on centrosome biogenesis or on spindle assembly in vivo. The RHAMM mutants exhibit misorientation of the division plane of neuroprogenitors, without affecting the division rate of these cells, resulting against expectation in megalencephaly associated with cerebral cortex thickening, cerebellum enlargement, and premature cerebellum differentiation. We conclude that RHAMM associates with the spindle of neuroprogenitor cells via its centrosome-targeting domain, where it regulates differentiation in the developing brain by orienting the spindle., Graphical Abstract, Highlights • Cerebral cortex apical progenitors (APs) and cerebellar GCPs exhibit planar division • Spindle-associated RHAMM orients the division plane of these neuroprogenitors • Excessive apicobasal division of APs causes cerebral cortex thickening • Decreased planar division of GCPs causes cerebellum enlargement, Ploubidou and colleagues assess the impact of misorientation of the mitotic spindle of neuronal progenitor cells on brain development. They employ a spindle misorientation model expressing truncated RHAMM, exhibiting no centrosome biogenesis or spindle assembly defects in vivo. The data demonstrate that spindle misorientation in cerebellum and cerebrum neuroprogenitors results in megalencephaly and that RHAMM regulates differentiation in the developing brain.

KRAS is the most frequently mutated driver of pancreatic, colorectal, and non–small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF–MEK–ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. Significance: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D. See related commentary by Zhao et al., p. 17. This article is highlighted in the In This Issue feature, p. 1

The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.

KAT6A and KAT6B are histone acetyl transferase (HAT) that are controlling gene expression by transferring acetyl groups to histones. Yu et al (Oncogene, 2017 36, 2910-2918) have shown that KAT6A has a role in breast cancer development by activating the ERα promoter and was found frequently amplified in 11% and is overexpressed in 15% of breast cancers. Altogether, these data support KAT6A as an attractive target in estrogen receptor driven breast cancer. KAT6B is a close homolog that shares 91% identity in the acetyl coA binding site. While the prevalence of gene amplification for KAT6B in breast cancer is only 1-2%, it is often overexpressed. In this study, we are exploring how KAT6A and KAT6B are contributing to ERα expression and the growth of KAT6A amplified breast cancer cell lines. To decipher each protein's role, we have designed specific siRNA against KAT6A and KAT6B and we tested their action either individually or in combination in the KAT6A gene amplified breast cancer cell lines T-47D, CAM-1 and ZR-75. Following treatment of T-47D, CAMA-1 and ZR75 cell lines with KAT6A or B siRNA we observed a decrease of ERα gene and protein expression for all siRNAs. The effect was more pronounced when using KAT6A over KAT6B and the effect of both siRNA A/B were additive. Phenotypically, the impact on cell line proliferation by applying clonogenic and incucyte assays, mirrors what is observed on ERα gene and protein expression. The effect on cellular proliferation of KAT6A is more prominent than KAT6B silencing, while the combined KAT6A + KAT6B silencing being more impactful than KAT6A silencing alone. In conclusion, our data show that KAT6A and KAT6B share similar function in the control of ER gene and protein expression. This study suggests that KAT6A and KAT6B may be paralogs, one compensating for the loss of the other. This translates into a more pronounced antiproliferative effect when both genes are silenced concomitantly. Citation Format: Isabelle Meaux, Dimitri Gorge-Bernat, Angele Paz, Catherine Geslin, Laurent Debussche, Jürgen Moll, Christophe Henry. Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2125.

Over the past years, the HIPPO tumor suppressor pathway and its downstream effector Yes1-associated transcriptional regulator (YAP1) present increasing interest in cancer research. The YAP1-TEAD complex regulates transcription for cell proliferation, survival, plasticity, and migration. Aberrant activation of YAP1 has been reported for multiple different cancer types. To estimate the proportion of cases where YAP1/TEAD-dependent transcription may be responsible for tumor development or evolution and evaluate the pharmacodynamics of novel TEAD-inhibitors, we have identified a transcriptional signature of YAP1 activity. The signature comprises 500 genes, positive and negative downstream effectors at about equal proportions, selected by differential expression analyses from 10 published datasets reporting, in total, 19 YAP1-activation and 18 YAP1-inhibition experiments. YAP1/TEAD-dependent transcription was scored in single samples as the difference Rp - Rn, where Rp is the mean fractional rank of the positive YAP1-effectors, and Rn, that of the negative effectors. We so scored, and called, YAP1 activation in normal tissues (GTEx collection; N~9000), in primary, recurrent, or metastatic tumors (TCGA; N~10000), and in cell lines (CCLE; N~1000) for the selection of models. We further applied the signature to follow the degree of YAP1 inhibition in cell and tumor models either after genetic knockdown of HIPPO-YAP1 pathway components or by pharmacologic inhibition using small-molecule TEAD-inhibitors. Our data confirm the robustness of the YAP1-transcription signature as a biomarker for both, tumor indication selection and pharmacodynamics of YAP1-TEAD activity. Citation Format: Emmanuel Spanakis, Loreley Calvet, Odette Dos Santos, Colette Dib, Sukhvinder Sidhu, Jurgen Moll, Laurent Debussche, Jack Pollard, Iris Valtingojer. A transcriptomic signature for measuring YAP1 activity in patient samples and tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2161.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM—a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382–95. ©2016 AACR.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion. The heterogeneity of breast cancer subtypes can be captured using organoid cultures that can facilitate drug screens that corroborate with patient responses.

Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal IGF2 amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An in vitro screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. Mol Cancer Ther; 16(10); 2223–33. ©2017 AACR.

MDM2-p53 antagonists block the interaction between the Tumor Protein p53 and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2-p53 antagonists are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2-p53 antagonists are currently being evaluated in early clinical development. BI-907828 is a novel and potent MDM2-p53 antagonist with optimized drug-like properties that has shown efficacy in human tumor xenograft models at daily low oral dose as well as intermittent high dose schedules. Recent preclinical studies in syngeneic mouse models of cancer have demonstrated that BI-907828, apart from its direct tumor-targeting activity, also has immunomodulatory activity shown to contribute to efficacy. Single-agent BI-907828 induced anti-tumor immunological memory that controlled tumor growth in a re-challenge study. Moreover, a dual combination with an anti-mouse PD-1 checkpoint inhibitor resulted in synergistic efficacy in a syngeneic mouse model of cancer (AACR 2018, abstract 4866). Here we present data for the triple combination of BI-907828 with two checkpoint inhibitors. In syngeneic mouse tumor models (Colon-26 and B16-F10), the combination of BI-907828 with tool antibodies against mouse PD-1 and mouse LAG-3 shows high response rates of 50-90% with tumor regressions observed for even very large tumors. Moreover, efficacy of the triple combination is superior to each single agent and all dual combinations. An antibody-mediated depletion study suggests a contribution of CD8+ T cells but not of CD4+ T cells to full efficacy of the triple combination. FACS analysis of tumors isolated from Colon-26 tumor-bearing mice indicates that treatment with the triple combination leads to expansion of tumor-infiltrating CD8+ T cells. In summary, BI-907828 is a novel, potent, orally bioavailable MDM2-p53 antagonist that shows synergistic efficacy in a triple combination with antibodies targeting the immune checkpoints PD-1 and LAG-3 in syngeneic mouse models of cancer. BI-907828 is currently under evaluation in a Phase I clinical study (NCT03449381). Citation Format: Dorothea Rudolph, Ulrike Weyer-Czernilofsky, Markus Reschke, Martina Sykora, Jörg Rinnenthal, Sophia Blake, Gabriela Gremel, Andreas Wernitznig, Andreas Gollner, Norbert Kraut, Jürgen Moll. BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3197.

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells. Cancer Res; 70(24); 10255–64. ©2010 AACR.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept in cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development. While the tumor targeting activity of MDM2 inhibitors has been well documented, recent preclinical data with the tool compound Nutlin-3 have shown that p53 activation by local, intra-tumoral injections induces anti-tumor immunity (Cancer Res 2017, 77(9) 2292-2305). Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties that is suitable for intermittent dose schedules in syngeneic mouse models of cancer. Our data show that immune modulation contributes to efficacy of BI 907828 after oral administration in a Colon-26 syngeneic mouse model. Responding mice develop anti-tumor immunity that can control the tumor growth of a secondary tumor without any further treatment in a re-challenge study. A T cell depletion study shows a contribution of CD8+ T cells but not of CD4+ T cells to single-agent efficacy with BI 907828. Moreover, preclinical data in a Colon-26 syngeneic mouse model of cancer show synergistic efficacy for the combination of a PD-1 checkpoint inhibitor with BI 907828. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows synergistic efficacy in combination with a PD-1 checkpoint inhibitor in syngeneic mouse models of cancer. Citation Format: Dorothea Rudolph, Markus Reschke, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Andreas Gollner, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Kraut Norbert, Jürgen Moll. BI 907828: A novel, potent MDM2 inhibitor that induces antitumor immunologic memory and acts synergistically with an anti-PD-1 antibody in syngeneic mouse models of cancer [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 4866.

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [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 4558.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly, thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties including a reliable, dose-linear PK across species with good bioavailability after oral dosing that allows various dose schedules. Remarkably, a single oral dose of 2 mg/kg led to tumor regressions in a SJSA-1 xenograft model in all treated mice, as did treatment on a daily low dose schedule. Data on PK, PD and efficacy relationships in this xenograft model will be presented including dose-dependent induction of TP53 target genes and markers of apoptosis. Moreover, BI 907828 significantly prolonged the survival by more than 50 days with a daily oral dose of 2.5 mg/kg in a difficult to treat disseminated MOLM-13 AML model, as noted by profiling of a clinical-stage MDM2 inhibitor. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows excellent efficacy with daily low dose but also intermittent high dose schedules in preclinical models of cancer. Citation Format: Dorothea Rudolph, Andreas Gollner, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Jürgen Moll, Kraut Norbert. BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules [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 4868.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present pharmacokinetic data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties. Due to its high permeability, good physiological solubility and low systemic clearance, the compound shows a high bioavailability in mice, rats, dogs and minipigs. BI 907828 is not absorption-limited, even at multiples of the efficacious dose, and shows a reliable, dose-linear PK with low variability in AUC and Cmax across species. Based on a good PK-PD correlation with efficacy in preclinical models and a robust human PK prediction based on in vivo PK data across species, a low human efficacious dose is predicted. The PK properties of BI 907828 enable a variety of different clinical dosing schedules with the potential to improve the therapeutic index with respect to on-target safety. In summary, BI 907828 is a novel, potent, orally available MDM2 inhibitor with excellent PK properties and low estimated human dose suitable for high-dose intermittent dose schedules in the clinic. Citation Format: Joerg Rinnenthal, Dorothea Rudolph, Sophia Blake, Andreas Gollner, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jürgen Moll, Norbert Kraut, Darryl McConnell, Jens Quant. BI 907828: A highly potent MDM2 inhibitor with low human dose estimation, designed for high-dose intermittent schedules in the clinic [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 4865.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor involved in maintaining immunological tolerance via regulation of T-cell activation, proliferation and response. Ligand-mediated activation of LAG-3 negatively regulates T-cell activity that is thought to actively contribute to tumor immune evasion beyond the established programmed cell death-1 (PD-1) pathway. BI 754111, a humanized IgG4 monoclonal antibody (mAb) with high affinity against LAG-3, blocks the interaction between LAG-3 and MHC II. BI 754111 was characterized in a panel of binding, blocking and functional cell-based assays; safety assessment was done in cynomolgus monkeys. BI 754111 is not mouse cross-reactive; therefore a surrogate mLAG-3 antibody was used for in vivo mouse efficacy studies. The ability of BI 754111 to stimulate cytokine production by exhausted human T cells in vitro was tested in an autologous assay system with antigen specific memory T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of increasing amounts of BI 754111 or BI 754091 (anti-hPD-1 mAb) or a combination of increasing amounts of BI 754111 and a saturating dose of BI 754091. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on the surface. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure of T-cell activation. Monotherapy of BI 754111 moderately increased IFNγ secretion (average of 1.8 fold) compared to BI 754091 monotherapy (6.9-fold average increase). Combining BI 754111 with BI 754091 was synergistic and led to a 13.2-fold increase in IFNγ secretion. MC-38 tumor-bearing mice (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice expressing parts of the human instead of the murine extracellular domain of PD-1) were used to determine the in vivo activity of the anti-LAG3 and anti-PD-1 combination. A mouse tool antibody against mLAG-3 (IgG1 D265A) and BI 905725 (a mouse IgG1 D265A version of BI 754091, anti-hPD-1 mAb) were tested at a dose of 10 mg/kg in a q3or4d schedule as monotherapy or in combination. No anti-tumor activity was observed under mLAG-3 mAb treatment, whereas treatment with anti-PD-1 resulted in a median TGI of 100% and 4 out of 10 tumors showed a complete response. The combination of anti-PD-1 and anti-LAG-3 resulted in a median TGI of 103% and doubled the number of complete responses (8/10). BI 754111 binds to cynomolgus monkey LAG-3 with comparable affinity as to hLAG-3 thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754111 were well tolerated without adverse immune-related side effects. The above results describe synergistic effects upon combination of PD-1 and LAG-3 treatment thus supporting the ongoing clinical trial in which BI 754111 is being tested in combination with BI 754091 (NCT03156114). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. Characterization of the LAG-3 targeting antibody BI 754111 in monotherapy and in combination with the anti-PD-1 antibody BI 754091 [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 4547.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G2/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL–positive and –negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34+ cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.

Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine. [Cancer Res 2007;67(17):7987–90]

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

Background: Cyclin-dependent kinase 8 (CDK8) is part of the mediator complex that can either positively or negatively influence transcription. CDK8 is known to phosphorylate signal transducer and activator of transcription 1 (STAT1) at the position Ser727. STAT1 activity is regulated by JAK-mediated phosphorylation of tyrosine701 which leads to dimerization, nuclear translocation and IFN-γ induced phosphorylation mediated by CDK8. Introduction of an alanine mutation at the phosphorylation site STAT1-S727 results in enhanced NK cell cytotoxicity accompanied by increased levels of perforin and granzyme B (Putz et al. 2013). Method: Here we present the discovery and development of potent and selective CDK8 inhibitors guided by crystallography. The inhibitory effect of optimized compounds BI 9811 and BI 1347 on STAT1 phosphorylation and perforin release was investigated in the human NK cell line NK-92MI. Direct effects on cancer cells were furthermore analyzed in a broad panel of cell lines. The compound BI 1347 was profiled in vivo in the orthotopic B16-F10 melanoma mouse model. Results: Highly potent and selective CDK8 inhibitors were identified with an IC50 of below 10 nM in a biochemical kinase assay, which translated in a potent down regulation of the STAT1- Ser727 signal and in increased perforin and granzyme B secretion. BI 9811 and BI 1347 were highly selective for CDK8, as tested in a broad kinase panel and showed no cytotoxic activity on NK cells and most cancer cell lines, which distinguishes this compound class from published CDK8 inhibitors. A representative molecule out of this compound class demonstrated in vivo biomarker modulation and survival increase in the murine B16-F10 melanoma mouse model. Conclusion: We developed potent CDK8 inhibitors that show activation of NK cells that translates into biomarker modulation (pSTAT1Ser727) and in vivo efficacy. Citation Format: Marco H. Hofmann, Harald Engelhardt, Sebastian Carotta, Heribert Arnhof, Dirk Scharn, Marc Kerenyi, Moritz Mayer, Gerhard Gmaschitz, Georg Egger, Christian Engelhardt, Michael Sanderson, Maria A. Impagnatiello, Renate Schnitzer, Mark Pearson, Darryl McConnell, Norbert Kraut, Jürgen Moll. Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4630. doi:10.1158/1538-7445.AM2017-4630

Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGs), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

The glycosaminoglycan hyaluronate (HA) is part of the extracellular environment in bone marrow. We show here that HA activates signal transduction cascades important for hemopoiesis. In myeloid and lymphoid long-term bone marrow cultures (LTBMC), treatment with hyaluronidase (HA’ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown. HA induces bone marrow macrophages to secrete IL-1β (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal transduction pathways: CD44 links to a pathway activating p38 protein kinase while the other yet unknown receptor induces Erk activity. There was no difference of the effect of HA and HA’ase on hematopoiesis in LTBMC and on cytokine production by macrophages in CD44-deficient mice compared with wild-type mice, indicating that the CD44 hyaluronate receptor and its signal transduction can be compensated for. Our data suggest a regulatory role for the extracellular matrix component HA in hematopoiesis and show the induction of signal transduction by HA receptors.