HOXB13 and its downstream targets in ovarian cancer progression

HOX geni se povezuju s različitim aspektima neoplastične progresije, budući da ektotopsko izražavanje nekih HOX gena promovira staničnu transformaciju in vitro i tumorogenezu in vivo. Pojačano izražavanje gena HOXB13 primijećeno je kod visokog postotka tumora dojke i jajnika te se povezuje s izrazito agresivnim kliničkim tijekom. Početni cilj ovog istraživanja je ispitati utjecaj ektotopskog izražavanja gena HOXB13 u genetički definiranim modelnim staničnim linijama karcinoma jajnika. Western analizama posredno je identificirano nekoliko gena čije je izražavanje u visokoj pozitivnoj ili negativnoj korelaciji s izražavanjem gena HOXB13. Među ovim genima, posebno se istaknuo klasterin, gen čiji se proteinski produkt (CLU) javlja u dvije izoforme. sCLU posjeduje signalnu sekvencu za sekreciju van stanice te mu se pripisuje citoprotektivna uloga, dok je nCLU lokaliziran u citoplazmi, a u slučaju oštećenja stanice odlazi u jezgru gdje obavlja proapoptotsku ulogu. Kao dio projekta identifikacije novih molekularnih meta koje bi bile podložne terapeutskoj intervenciji u karcinomu jajnika, ovaj rad opisuje proces dobivanja dviju novih staničnih linija koje predstavljaju primjere međusobno disjunktnih uloga klasterina. Dobivene modelne linije analizirane su in vitro i in vivo, te je dokazano stabilno ektotopsko izražavanje obiju klasterinskih izoformi.The HOX genes have been implicated in different aspects of the oncogenic process, as ectopic expression of some HOX genes promotes cellular transformation in vitro and tumorigenesis in vivo. The observation that HOXB13 is expressed in a high percentage of breast and ovarian cancer cell lines and tumors, and that the expression of HOXB13 is associated with a more aggressive clinical course, led us to investigate the ectopic expression of HOXB13 in genetically defined ovarian cancer cell lines. We report identification of several putative HOXB13 targets among which clusterin takes the most prominent role. Clusterin is present in two distinct isoforms, as secreted clusterin (sCLU) and as nuclear clusterin (nCLU). While the former is associated with cell survival, the latter is pro-apoptotic. As a part of the project aiming to identify new therapeutic targets for ovarian cancer, here we describe the generation of clusterinoverexpressing cell lines, representing two contradictory functions of clusterin. These cell lines have subsequently been analyzed using both in vitro and in vivo assays. Consequently, stable ectopic expression of both clusterin isoforms has been demonstrated

Immunoassay for quantification of antigen-specific IgG fucosylation

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021

HOXB13 and its downstream targets in ovarian cancer progression

HOX geni se povezuju s različitim aspektima neoplastične progresije, budući da ektotopsko izražavanje nekih HOX gena promovira staničnu transformaciju in vitro i tumorogenezu in vivo. Pojačano izražavanje gena HOXB13 primijećeno je kod visokog postotka tumora dojke i jajnika te se povezuje s izrazito agresivnim kliničkim tijekom. Početni cilj ovog istraživanja je ispitati utjecaj ektotopskog izražavanja gena HOXB13 u genetički definiranim modelnim staničnim linijama karcinoma jajnika. Western analizama posredno je identificirano nekoliko gena čije je izražavanje u visokoj pozitivnoj ili negativnoj korelaciji s izražavanjem gena HOXB13. Među ovim genima, posebno se istaknuo klasterin, gen čiji se proteinski produkt (CLU) javlja u dvije izoforme. sCLU posjeduje signalnu sekvencu za sekreciju van stanice te mu se pripisuje citoprotektivna uloga, dok je nCLU lokaliziran u citoplazmi, a u slučaju oštećenja stanice odlazi u jezgru gdje obavlja proapoptotsku ulogu. Kao dio projekta identifikacije novih molekularnih meta koje bi bile podložne terapeutskoj intervenciji u karcinomu jajnika, ovaj rad opisuje proces dobivanja dviju novih staničnih linija koje predstavljaju primjere međusobno disjunktnih uloga klasterina. Dobivene modelne linije analizirane su in vitro i in vivo, te je dokazano stabilno ektotopsko izražavanje obiju klasterinskih izoformi.The HOX genes have been implicated in different aspects of the oncogenic process, as ectopic expression of some HOX genes promotes cellular transformation in vitro and tumorigenesis in vivo. The observation that HOXB13 is expressed in a high percentage of breast and ovarian cancer cell lines and tumors, and that the expression of HOXB13 is associated with a more aggressive clinical course, led us to investigate the ectopic expression of HOXB13 in genetically defined ovarian cancer cell lines. We report identification of several putative HOXB13 targets among which clusterin takes the most prominent role. Clusterin is present in two distinct isoforms, as secreted clusterin (sCLU) and as nuclear clusterin (nCLU). While the former is associated with cell survival, the latter is pro-apoptotic. As a part of the project aiming to identify new therapeutic targets for ovarian cancer, here we describe the generation of clusterinoverexpressing cell lines, representing two contradictory functions of clusterin. These cell lines have subsequently been analyzed using both in vitro and in vivo assays. Consequently, stable ectopic expression of both clusterin isoforms has been demonstrated

From resistance to sensitivity and back – the full circle of resisting the inescapable

Cancer is a genetic disease characterized by the unrestrained proliferation of malignantly transformed cells. In this thesis we study the mechanisms of resistance to targeted therapeutics, drugs that target specifically the protein products of mutated genes “driving” the progression of cancer, or signaling pathways that are hyper-activated by cancer-promoting mutations. Some of these agents have made a significant clinical success, especially in terms of prolonging progression-free survival, but with limited improvements in overall survival due to the emergence of resistance. Chapter 1 gives an overview of main strategies used to overcome the development of resistance to targeted agents, such as drug combination treatments based on genetic concepts of synthetic lethality and collateral dependency. Chapter 2 presents the discovery of a synthetic lethal drug combination of FGFR and PI3K inhibitors in FGFR-driven lung and bladder cancer. This finding was based on a functional genetic screen, an unbiased approach designed to find enhancers of sensitivity to FGFR inhibitors. We validate our findings in vitro, using both genetic and pharmacological approach, and in vivo, using xenograft mouse models. We also uncover the molecular mechanism underlying this synergy, showing that the inhibition of FGFR causes a rapid feedback activation of the receptor tyrosine kinases (RTKs) EGFR and HER3. This finding gives a solid rationale for clinical testing of FGFR inhibitors in combination with PI3K inhibitors in cancers driven by the genetic activation of the FGFR genes. Chapter 3 starts with a goal to find synthetic lethal interactions specific to mutant RAS. We describe the results of genome-wide genetic screens in yeast, and validate them in KRAS mutant colorectal cancer cells. We find that the loss of the endoplasmic reticulum (ER) stress sensor ERN1 does not affect growth, but sensitizes to MEK inhibition. Next, we uncover the mechanistic connection between ERN1 and the MAPK pathway and establish the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. This finding contributes to explaining the resistance of KRAS mutant tumor cells to MEK inhibitor treatment. Chapter 4 uncovers the unexpected connection between the osteogenic master regulator transcription factor RUNX2 and its cofactor CBFB, with the MAPK pathway. We first show that the loss of RUNX2 or CBFB can confer MEK inhibitor resistance in colorectal cancer cells. Mechanistically, we find that the inactivation of these genes results in activation of multiple RTKs which is mirrored by the high SHP2 phosphatase activity. Next, we find that high SHP2 activity has a causal role to loss of RUNX2-induced MEK inhibitor resistance. Finally, we find that SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout KRAS mutant colorectal cancer cells. Chapter 5 gives an overview of the recently reported clinical studies on PI3K inhibitor buparlisib used in Chapter 2. This is relevant for understanding future perspectives of our findings. Finally, I discuss the limitations of the synthetic lethality as a concept, and give examples of RAS synthetic lethality studies, discussing how they complement the work described in Chapters 3 and 4

From resistance to sensitivity and back – the full circle of resisting the inescapable

Cancer is a genetic disease characterized by the unrestrained proliferation of malignantly transformed cells. In this thesis we study the mechanisms of resistance to targeted therapeutics, drugs that target specifically the protein products of mutated genes “driving” the progression of cancer, or signaling pathways that are hyper-activated by cancer-promoting mutations. Some of these agents have made a significant clinical success, especially in terms of prolonging progression-free survival, but with limited improvements in overall survival due to the emergence of resistance. Chapter 1 gives an overview of main strategies used to overcome the development of resistance to targeted agents, such as drug combination treatments based on genetic concepts of synthetic lethality and collateral dependency. Chapter 2 presents the discovery of a synthetic lethal drug combination of FGFR and PI3K inhibitors in FGFR-driven lung and bladder cancer. This finding was based on a functional genetic screen, an unbiased approach designed to find enhancers of sensitivity to FGFR inhibitors. We validate our findings in vitro, using both genetic and pharmacological approach, and in vivo, using xenograft mouse models. We also uncover the molecular mechanism underlying this synergy, showing that the inhibition of FGFR causes a rapid feedback activation of the receptor tyrosine kinases (RTKs) EGFR and HER3. This finding gives a solid rationale for clinical testing of FGFR inhibitors in combination with PI3K inhibitors in cancers driven by the genetic activation of the FGFR genes. Chapter 3 starts with a goal to find synthetic lethal interactions specific to mutant RAS. We describe the results of genome-wide genetic screens in yeast, and validate them in KRAS mutant colorectal cancer cells. We find that the loss of the endoplasmic reticulum (ER) stress sensor ERN1 does not affect growth, but sensitizes to MEK inhibition. Next, we uncover the mechanistic connection between ERN1 and the MAPK pathway and establish the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. This finding contributes to explaining the resistance of KRAS mutant tumor cells to MEK inhibitor treatment. Chapter 4 uncovers the unexpected connection between the osteogenic master regulator transcription factor RUNX2 and its cofactor CBFB, with the MAPK pathway. We first show that the loss of RUNX2 or CBFB can confer MEK inhibitor resistance in colorectal cancer cells. Mechanistically, we find that the inactivation of these genes results in activation of multiple RTKs which is mirrored by the high SHP2 phosphatase activity. Next, we find that high SHP2 activity has a causal role to loss of RUNX2-induced MEK inhibitor resistance. Finally, we find that SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout KRAS mutant colorectal cancer cells. Chapter 5 gives an overview of the recently reported clinical studies on PI3K inhibitor buparlisib used in Chapter 2. This is relevant for understanding future perspectives of our findings. Finally, I discuss the limitations of the synthetic lethality as a concept, and give examples of RAS synthetic lethality studies, discussing how they complement the work described in Chapters 3 and 4

RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35-45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors

A role for the unfolded protein response stress sensor ERN1 in regulating the response to MEK inhibitors in KRAS mutant colon cancers

Abstract Background Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. Methods We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy. Our method used selective ploidy ablation, which enables replication of cancer-specific gene expression changes in the yeast gene disruption library. Second, we used a genome-wide CRISPR/Cas9-based genetic screen in KRAS mutant human colon cancer cells to understand the mechanistic connection between the synthetic lethal interaction discovered in yeast and downstream RAS signaling in human cells. Results We identify loss of the endoplasmic reticulum (ER) stress sensor IRE1 as synthetic lethal with activated RAS mutants in yeast. In KRAS mutant colorectal cancer cell lines, genetic ablation of the human ortholog of IRE1, ERN1, does not affect growth but sensitizes to MEK inhibition. However, an ERN1 kinase inhibitor failed to show synergy with MEK inhibition, suggesting that a non-kinase function of ERN1 confers MEK inhibitor resistance. To investigate how ERN1 modulates MEK inhibitor responses, we performed genetic screens in ERN1 knockout KRAS mutant colon cancer cells to identify genes whose inactivation confers resistance to MEK inhibition. This genetic screen identified multiple negative regulators of JUN N-terminal kinase (JNK) /JUN signaling. Consistently, compounds targeting JNK/MAPK8 or TAK1/MAP3K7, which relay signals from ERN1 to JUN, display synergy with MEK inhibition. Conclusions We identify the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. The notion that multiple signaling pathways can activate JUN may explain why KRAS mutant tumor cells are traditionally seen as highly refractory to MEK inhibitor therapy. Our findings emphasize the need for the development of new therapeutics targeting JUN activating kinases, TAK1 and JNK, to sensitize KRAS mutant cancer cells to MEK inhibitors

Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines

Summary: IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps