Immunomodulatory drugs (IMiDs), such as thalidomide and its derivatives lenalidomide and pomalidomide, are key treatment modalities for hematologic malignancies, particularly multiple myeloma (MM) ...and del(5q) myelodysplastic syndrome (MDS). Cereblon (CRBN), a substrate receptor of the CRL4 ubiquitin ligase complex, is the primary target by which IMiDs mediate anticancer and teratogenic effects. Here we identify a ubiquitin-independent physiological chaperone-like function of CRBN that promotes maturation of the basigin (BSG; also known as CD147) and solute carrier family 16 member 1 (SLC16A1; also known as MCT1) proteins. This process allows for the formation and activation of the CD147-MCT1 transmembrane complex, which promotes various biological functions, including angiogenesis, proliferation, invasion and lactate export. We found that IMiDs outcompete CRBN for binding to CD147 and MCT1, leading to destabilization of the CD147-MCT1 complex. Accordingly, IMiD-sensitive MM cells lose CD147 and MCT1 expression after being exposed to IMiDs, whereas IMiD-resistant cells retain their expression. Furthermore, del(5q) MDS cells have elevated CD147 expression, which is attenuated after IMiD treatment. Finally, we show that BSG (CD147) knockdown phenocopies the teratogenic effects of thalidomide exposure in zebrafish. These findings provide a common mechanistic framework to explain both the teratogenic and pleiotropic antitumor effects of IMiDs.
Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, ...we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
The azanucleosides azacitidine and decitabine are currently used for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in patients not only eligible for intensive ...chemotherapy but are also being explored in other hematologic and solid cancers. Based on their capacity to interfere with the DNA methylation machinery, these drugs are also referred to as hypomethylating agents (HMAs). As DNA methylation contributes to epigenetic regulation, azanucleosides are further considered to be among the first true "epigenetic drugs" that have reached clinical application. However, intriguing new evidence suggests that DNA hypomethylation is not the only mechanism of action for these drugs. This review summarizes the experience from more than 10 years of clinical practice with azanucleosides and discusses their molecular actions, including several not related to DNA methylation. A particular focus is placed on possible causes of primary and acquired resistances to azanucleoside treatment. We highlight current limitations for the success and durability of azanucleoside-based therapy and illustrate that a better understanding of the molecular determinants of drug response holds great potential to overcome resistance.
The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular ...communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
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•CRBN functions as a transmembrane protein-specific co-chaperone of HSP90•Disruption of CRBN-HSP90 interaction determines the anti-tumor activity of IMiDs•The CD98hc/LAT1 complex is a central target of IMiDs in multiple myeloma•CD98hc-Anticalin is a theranostic tool in multiple myeloma
Heider et al. investigate the molecular function of immunomodulatory drugs (IMiDs) and describe their target, CRBN, as a transmembrane protein (TP)-specific co-chaperone of the HSP90-AHA1 axis. By disrupting CRBN-HSP90 interaction, IMiDs lead to destabilization of various TPs as CD98hc/LAT1, which serve as therapeutic targets in multiple myeloma.
Genetic lesions affecting epigenetic regulators are frequent in myelodysplastic syndromes (MDS). Polycomb proteins are key epigenetic regulators of differentiation and stemness that act as two ...multimeric complexes termed polycomb repressive complexes 1 and 2, PRC1 and PRC2, respectively. While components and regulators of PRC2 such as ASXL1 and EZH2 are frequently mutated in MDS and AML, little is known about the role of PRC1. To analyze the role of PRC1, we have taken a functional approach testing PRC1 components in loss- and gain-of-function experiments that we found overexpressed in advanced MDS patients or dynamically expressed during normal hematopoiesis. This approach allowed us to identify the enzymatically active component RING1A as the key PRC1 component in hematopoietic stem cells and MDS. Specifically, we found that RING1A is expressed in CD34
bone marrow progenitor cells and further overexpressed in high-risk MDS patients. Knockdown of RING1A in an MDS-derived AML cell line facilitated spontaneous and retinoic acid-induced differentiation. Similarly, inactivation of RING1A in primary CD34
cells augmented erythroid differentiation. Treatment with a small compound RING1 inhibitor reduced the colony forming capacity of CD34
cells from MDS patients and healthy controls. In MDS patients higher RING1A expression associated with an increased number of dysplastic lineages and blasts. Our data suggests that RING1A is deregulated in MDS and plays a role in the erythroid development defect.
Effectively targeting leukemia-initiating cells (LIC) in
-ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing ...to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates
-ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show
-ITD occurs within the most primitive Lin
/CD33
/CD45
/CD34
CD38
LIC compartment. Crenolanib alone could not target
-ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC
and severely impaired engraftment in NSG mice. Strikingly,
-mutated samples harboring
mutations were completely resistant to crenolanib whereas neither
nor
mutations influenced response. Conversely, primary AML LIC harboring either
or
mutations did not show increased sensitivity to AZA. In summary, resistance of
-ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of
-ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.
Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor ...cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo–expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.
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Introduction:
Cereblon (CRBN) is the target for immunomodulatory drugs (IMiDs) such as thalidomide and its derivatives lenalidomide and pomalidomide, which are key therapeutics for hematologic ...malignancies such as multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). We have previously described a ubiquitin-independent chaperone-like function of CRBN, which stabilizes the transmembrane proteins CD147 and MCT1. IMiDs interfere with this chaperone-like function of CRBN in a competitive manner to mediate both their anti-tumor and their teratotoxic effects (Eichner et al. Nature Medicine 2016). So far, the underlying mechanisms of transmembrane protein maturation, the global impact of CRBN on the cell surface proteome and the precise molecular mechanism of IMiDs, especially their clinically well-established synergy with proteasomal inhibitors remain unclear.
Methods:
Novel CRBN-clients were identified by cross-validation of the CRBN-interactome with a cell surface proteomic screen. Various molecular and cell biological methods including immunofluorescence, flow cytometry, immunoprecipitations, GST-pulldowns, amino acid transport and proliferation assays were used to decipher underlying mechanisms. In vitro assays and in vivo xenograft experiments were performed using MM cell lines and patient-derived CD138+ MM cells. 18FDG- and 18FET-PET was used for imaging of xenografted tumors.
Results:
Our unbiased screening approaches imply a global role of CRBN in transmembrane protein maturation. In particular, we identify the amino acid transporter LAT1 and its functional subunit CD98hc as novel CRBN client proteins, which are frequently overexpressed in MM to drive cell proliferation. CD98hc/LAT1 become destabilized and inactivated upon IMiD treatment, which attenuates MM cell proliferation, tumor formation and perturbs the uptake of essential amino acids, thereby further linking IMiD-activity to tumor-metabolism. CD98hc/LAT1 destabilization only occurs in IMiD-sensitive patients and cell lines, thus being a potential biomarker to predict IMiD-response. Moreover, inhibition of LAT1 is cytotoxic in both IMiD-sensitive and -resistant cells, which makes it an attractive therapeutic option for IMiD-resistant and -refractory patients. Mechanistically, we show CRBN to function as a new selective co-chaperone of HSP90, which facilitates transmembrane protein maturation in a ubiquitin-independent way, which is impaired by IMiD-treatment.
Conclusion:
We establish CRBN as a transmembrane protein-specific co-chaperone for HSP90 and identify modulation of the CRBN-CD98hc/LAT1 axis as crucial means by which IMiDs mediate their anti-tumor activity. Notably, we specify CD98hc/LAT1 as valuable biomarkers for IMiD-response and druggable targets for IMiD-resistant and -refractory MM patients and beyond. Furthermore, this ubiquitin-independent mechanism solves the paradox regarding the well documented synergistic anti-myeloma activity of IMiDs and proteasomal inhibitors.
Götze:AbbVie: Membership on an entity's Board of Directors or advisory committees. Bassermann:Celgene: Consultancy, Research Funding.
Background: Hematopoietic stem/progenitor cells (HSPC) are highly dependent on interaction with the specific bone marrow microenvironment (niche) which supports their survival and directs their ...differentiation. Increasing evidence suggests that the niche plays a central role in the development of clonal stem cell disorders such as myelodysplastic syndromes (MDS). Malignant clonal HSPC in MDS have been shown to reprogram mesenchymal stromal cells (MSC) to promote disease progression. In turn, the altered stromal microenvironment in MDS may directly impair healthy hematopoiesis. Accordingly, MSC isolated from MDS patients have shown phenotypical and functional abnormalities, which were not observed in patients successfully treated with azacitidine (AZA), currently the standard of care for higher-risk MDS patients. Although AZA is a potent inhibitor of DNA methyltransferases and is thus considered a hypomethylating agent, the exact anti-leukemic mechanisms are not understood and a clear correlation between DNA demethylation and response to AZA has not been demonstrated thus far. Hence, we looked beyond the current focus of AZA effects on clonal hematopoiesis and hypothesized that AZA may also act through direct modulation of the MSC compartment. To answer this question, we examined primary MSC treated in vitro with AZA as well as the functional interaction between MSC and HSPC after AZA treatment.
Methods: Primary MSC were isolated from bone marrow of untreated MDS patients or age-matched healthy controls, expanded by plastic adherence, cultured until confluency under defined conditions and used for up to 4 passages. Identity of MSC was confirmed by presence of characteristic cell surface markers CD73, CD271, CD105 and CD90 and absence of CD45 and CD34 by flow cytometry. MSC were differentiated into the adipogenic or osteogenic lineage after in vitro treatment with AZA (10 uM). Co-culture experiments with CD34+ HSPC from MDS patients or healthy controls were performed on AZA-treated or untreated MDS-MSC or healthy MSC, respectively. After four days, CD34+ cells were harvested and assessed by colony-forming unit assays in methylcellulose. Single colonies were genotyped for MDS-associated mutations known to be present in the bone marrow sample. Each colony was genotyped for one or two known MDS-associated mutations and an input patient specimen was used as a control. Finally, RNA sequencing was performed on a mesenchymal stromal cell line (EL08-1D2), either untreated or treated with AZA for 4 days in vitro to uncover pathways affected by AZA in stromal cells.
Results: MDS-MSC showed a decreased proliferative capacity as well as markedly impaired differentiation into the osteoblastic lineage compared to healthy MSC. Treatment with AZA had no effect on cell surface marker expression or viability of either healthy or MDS-MSC. AZA treatment significantly reduced the ability of MDS-MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was severely impaired with or without AZA treatment. In contrast, osteogenic differentiation of healthy MSC was enhanced after AZA treatment. In co-culture experiments pretreatment with AZA significantly improved stromal support of healthy CD34+ HSPC while malignant MDS-HSPC were suppressed. RNA sequencing analysis revealed differential gene expression in AZA-treated vs untreated stroma with significantly more genes upregulated in AZA-treated samples. Specific changes in pathways involved in ECM-receptor interaction, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction and metabolic pathways in AZA treated EL08-1D2 stromal cells were determined and validated in primary MDS MSC samples.
Conclusion: We show here that AZA directly modulates altered MSC function in MDS, thus influencing interaction with HSPC and leading to suppression of malignant hematopoiesis. Our data suggest an additional mode of action by which AZA exerts its therapeutic activity. To our knowledge this analysis is the first to examine the direct effect of AZA treatment on MSC.
Götze:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Abbvie: Honoraria.
Background: Effectively targeting the oncogenic mutation FLT3-ITD remains a crucial goal in acute myeloid leukemia (AML) therapy. Thus far, tyrosine kinase inhibitors (TKI) have not been able to ...eradicate the earliest leukemia-initiating cells (LIC) in FLT3-ITD+ AML, which are thought to be responsible for the frequent relapses seen in this disease. We have previously shown that LIC in FLT3-ITD+ AML persist during treatment with first-generation TKI despite effective inhibition of FLT3 phosphorylation owing to their selective protection by niche cells (Parmar et al, Cancer Res 2011). Hence, novel strategies to disrupt the protective interaction of stroma with FLT3-ITD+ LIC are urgently needed. Here, we asked whether stromal resistance of FLT3-ITD+ LIC can be overcome by the next generation TKI crenolanib alone or in combination with the hypomethylating agent azacitidine (AZA).
Methods: The efficacy of crenolanib alone or in combination with AZA was assessed in the human FLT3-ITD+ cell lines MV4-11 and MOLM13 and FLT3-ITD transfected BaF3 cells as well as in primary human FLT3-ITD+ AML bone marrow samples. Cells were cultured in suspension or on the mesenchymal stromal cell line EL08-1D2, which mimics the bone marrow niche and maintains LIC in vitro (Parmar et al, Cancer Res 2011). Cultures were treated with DMSO, crenolanib and/or AZA for defined periods. Apoptosis, cell cycle and differentiation of AML cells were analyzed by flow cytometry. Clonogenic capacity and frequency of primitive FLT3-ITD+ stem/progenitors were probed by standard CFU and LTC assays. Engraftment potential of FLT3-ITD+ patient-derived xenograft (PDX) AML cells after treatment with crenolani, AZA or the combination thereof was assessed in the NSG xenograft model (Vick et al, PlosOne 2015). Treatment-induced alterations in FLT3-ITD downstream signaling were investigated by western blots.
Results: Crenolanib effectively inhibited FLT3 and STAT5 phosphorylation in FLT3-ITD+ cells in suspension as well as in EL08-1D2 supported co-cultures whereas AZA had no effect on FLT3 signaling pathways. Monotherapy with crenolanib but not AZA effectively induced apoptosis and inhibited growth of FLT3-ITD+ cell lines. Primary CD34+ FLT3-ITD+ progenitor cells were also highly susceptible to inhibition by crenolanib as a single agent. However, crenolanib was completely unable to eradicate primitive CD34+ FLT3-ITD+ LIC when protected by niche cells as assessed by CFU and LTC assays as well as xenotransplantation into NSG mice. In contrast, the combination of crenolanib and AZA resulted in efficient apoptosis and dramatically impaired clonogenic capacity of FLT3-ITD+ LIC even in the presence of stroma. Pretreatment of EL08-1D2 cells with AZA before co-culture with AML cells did not influence stromal protection against crenolanib. Furthermore, soluble stromal factors did not account for TKI resistance. Successful targeting of stromally protected FLT3-ITD+ LIC by the combination of AZA and crenolanib is currently being confirmed by xenotransplantation in NSG mice.
Conclusion: The combination of azacitidine and the selective next generation FLT3 kinase inhibitor crenolanib is a novel promising treatment regimen to overcome niche protection and selectively target LIC in FLT3-ITD+ AML. We hypothesize that the mechanism of action involves loss of quiescence in FLT3-ITD+ LIC leading to increased susceptibility towards TKI as well as induction of differentiation by AZA. In depth analysis of involved signaling pathways and phenotypic alterations in LIC are ongoing.
Keller:Pfizer: Consultancy; Roche: Consultancy, Honoraria. Götze:Novartis: Honoraria; Celgene Corp.: Honoraria.
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