Periodic Reporting for period 4 - BCM-UPS (Dissecting the role of the ubiquitin proteasome system in the pathogenesis and therapy of B-cell malignancies)
Período documentado: 2021-03-01 hasta 2022-08-31
The previous clinical success of proteasome inhibitors (e.g. Bortezomib, Carfilzomib) in the treatment of MM and MCL has highlighted the importance of the ubiquitin-proteasome system in these diseases and cancer in general. Moreover, immunomodulatory drugs (IMiDs) like thalidomide and its derivatives Lenalidomide and Pomalidomide which demonstrate high efficacy in newly diagnosed and relapsed/refractory MM and MCL, exert some anti-tumor activity by targeting the UPS. Therefore, the identification of disease-specific aberrant pathways of the UPS would be predicted to significantly contribute to a better understanding of these diseases, and define target structures whose inhibition may lead to new, more specific therapies.
Work within this project mechanistically specified different members of the ubiquitin system such as the deubiquitylases OTUD6B, USP9X and the E3 ligase FBXL13 as central vulnerabilities in MM and other B cell malignancies such as MCL and diffuse large B cell lymphoma (DLBCL). At the same time, these ubiquitin editing enzymes were characterized as highly attractive and amenable targets for future more specific treatment strategies as well as biomarkers for disease progression. In a further effort, the molecular mode of action of immunomodulatory drugs (IMiDs) such as Thalidomide, Lenalidomide and Pomalidomide was solved. These drugs are currently approved therapeutic cornerstones of B cell malignancies such as multiple myeloma and mantle cell lymphoma.
Work within this project mechanistically specified different members of the ubiquitin system such as the deubiquitylases OTUD6B, USP9X and the E3 ligase FBXL13 as central vulnerabilities in MM and other B cell malignancies such as MCL and diffuse large B cell lymphoma (DLBCL). At the same time, these ubiquitin editing enzymes were characterized as highly attractive and amenable targets for future more specific treatment strategies as well as biomarkers for disease progression. In a further effort, the molecular mode of action of immunomodulatory drugs (IMiDs) such as Thalidomide, Lenalidomide and Pomalidomide was solved. These drugs are currently approved therapeutic cornerstones of B cell malignancies such as multiple myeloma and mantle cell lymphoma.
Overview of results, Exploitation and Dissemination
Multiple Myeloma (MM) and Mantle cell lymphoma (MCL) each mark currently non-curable entities within the B cell malignancy family with a median survival of approximately 5 and 4 years, respectively. Therapies that inhibit the proteasome (PIs) or address certain ubiquitin ligases (IMiDs) have been successfully introduced into the therapy of MM and MCL, suggesting pathogenic and therapeutic roles of disease-specific deregulated ubiquitylation events, which have however remained largely elusive.
Within a candidate approach, we investigated the ubiquitin-specific protease 9X (USP9X), for which our initial analysis suggested a function within the mitotic spindle assembly checkpoint (SAC). We found that USP9X stabilizes the transcription factor Wilms tumor protein 1 (WT1) in mitosis, which in consequence promotes mitotic transcription of the oncogenic chemokine IL-8. This finding was intriguing, because mitosis has so far been considered transcriptionally silent. When overexpressed or aberrantly activated, USP9X can override the SAC and promote cell division. USP9X is significantly overexpressed in MM and MCL and therefore constitutes a highly attractive therapeutic target (Dietachmayr et al., Nat Commun 2021).
In a further candidate-based approach derived from systematic aCGH analyses in MCL, we investigated the F-box protein FBXL13. Here, we specified FBXL13 as a novel regulator of centrosome homeostasis and cell migration (Fung et al., 2018). More specifically, we found FBXL13 to target the centrosomal protein CEP192 for ubiquitin proteasomal degradation. CEP192 is a key factor in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. Our study revealed that induced FBXL13 expression downregulates centrosomal γ‐tubulin and reduces centrosomal microtubule nucleation capacity which results in an increased ability of cells to migrate. Our findings thus provide a frame-work from which to further investigate the function of FBXL13 in MCL, which are anticipated to provide novel insights into the role of aberrant centrosome homeostasis and cell motility in MCL pathogenesis and drug resistance (Fung et al., EMBO Rep. 2018).
To investigate the more global impact of the UPS on MM pathophysiology and maintenance, we performed unbiased CRISPR/Cas9-based functional genetic drop out screens using sgRNA libraries against human DUBs and SCF-family ubiquitin ligases to identify new vulnerabilities in this disease. This approach revealed various promising candidates, among which the deubiquitlylase OTUD6B was further characterized. We found that OTUD6B determines cell cycle progression of multiple myeloma cells by stabilizing the RNA binding protein LIN28B to attenuate microRNA-mediated restriction of MYC at G1/S. High OTUD6B expression associates with the MGUS-MM progression and is an adverse outcome of multiple myeloma. OTUD6B thus constitutes a highly attractive target in MM, also in terms of indirect MYC inactivation, which remains to be undruggable. Moreover, this new mechanism inks proteolytic ubiquitylation to mRNA biogenesis (Paulmann et al., EMBO J 2022). Current work focusses on the development of a specific OTUD6B inhibitor directed at the active cysteine site of this DUB.
Finally, we investigated the ubiquitin E3 ligase CRBN as to its role in mediating the molecular mode of action of immunomodulatory drugs (IMiDs) in MM. IMiDs such as Thalidomide, Lenalidomide and Pomalidomide are a central treatment pillar of MM but also MCL and myeloid malignancies such as del5q MDS. Surprisingly, we here identified a ubiquitin-independent function of CRBN by which this protein mediates quality control of central transmembrane proteins such as MCT1/CD147 and CD98/LAT1 that secure nutrient influx and waste efflux. IMiDs outcompete the quality control function of CRBN on these proteins with the consequence of their misfolding and removal by the unfolded protein response, eventually leading to cell death of MM cells (Heider et al., Mol Cell 2021). MCT1 expression was also specified as an independent negative prognostic marker for lenalidomide maintenance therapy within large MM patient cohorts, thus further underscoring the validity of this new mechanism of IMiD function (Stroh et al., Blood Adv 2022).
In addition to the above projects, various successful collaborations have been performed under the BCM-UPS funding and are specified in the “publications” section of this report.
Multiple Myeloma (MM) and Mantle cell lymphoma (MCL) each mark currently non-curable entities within the B cell malignancy family with a median survival of approximately 5 and 4 years, respectively. Therapies that inhibit the proteasome (PIs) or address certain ubiquitin ligases (IMiDs) have been successfully introduced into the therapy of MM and MCL, suggesting pathogenic and therapeutic roles of disease-specific deregulated ubiquitylation events, which have however remained largely elusive.
Within a candidate approach, we investigated the ubiquitin-specific protease 9X (USP9X), for which our initial analysis suggested a function within the mitotic spindle assembly checkpoint (SAC). We found that USP9X stabilizes the transcription factor Wilms tumor protein 1 (WT1) in mitosis, which in consequence promotes mitotic transcription of the oncogenic chemokine IL-8. This finding was intriguing, because mitosis has so far been considered transcriptionally silent. When overexpressed or aberrantly activated, USP9X can override the SAC and promote cell division. USP9X is significantly overexpressed in MM and MCL and therefore constitutes a highly attractive therapeutic target (Dietachmayr et al., Nat Commun 2021).
In a further candidate-based approach derived from systematic aCGH analyses in MCL, we investigated the F-box protein FBXL13. Here, we specified FBXL13 as a novel regulator of centrosome homeostasis and cell migration (Fung et al., 2018). More specifically, we found FBXL13 to target the centrosomal protein CEP192 for ubiquitin proteasomal degradation. CEP192 is a key factor in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. Our study revealed that induced FBXL13 expression downregulates centrosomal γ‐tubulin and reduces centrosomal microtubule nucleation capacity which results in an increased ability of cells to migrate. Our findings thus provide a frame-work from which to further investigate the function of FBXL13 in MCL, which are anticipated to provide novel insights into the role of aberrant centrosome homeostasis and cell motility in MCL pathogenesis and drug resistance (Fung et al., EMBO Rep. 2018).
To investigate the more global impact of the UPS on MM pathophysiology and maintenance, we performed unbiased CRISPR/Cas9-based functional genetic drop out screens using sgRNA libraries against human DUBs and SCF-family ubiquitin ligases to identify new vulnerabilities in this disease. This approach revealed various promising candidates, among which the deubiquitlylase OTUD6B was further characterized. We found that OTUD6B determines cell cycle progression of multiple myeloma cells by stabilizing the RNA binding protein LIN28B to attenuate microRNA-mediated restriction of MYC at G1/S. High OTUD6B expression associates with the MGUS-MM progression and is an adverse outcome of multiple myeloma. OTUD6B thus constitutes a highly attractive target in MM, also in terms of indirect MYC inactivation, which remains to be undruggable. Moreover, this new mechanism inks proteolytic ubiquitylation to mRNA biogenesis (Paulmann et al., EMBO J 2022). Current work focusses on the development of a specific OTUD6B inhibitor directed at the active cysteine site of this DUB.
Finally, we investigated the ubiquitin E3 ligase CRBN as to its role in mediating the molecular mode of action of immunomodulatory drugs (IMiDs) in MM. IMiDs such as Thalidomide, Lenalidomide and Pomalidomide are a central treatment pillar of MM but also MCL and myeloid malignancies such as del5q MDS. Surprisingly, we here identified a ubiquitin-independent function of CRBN by which this protein mediates quality control of central transmembrane proteins such as MCT1/CD147 and CD98/LAT1 that secure nutrient influx and waste efflux. IMiDs outcompete the quality control function of CRBN on these proteins with the consequence of their misfolding and removal by the unfolded protein response, eventually leading to cell death of MM cells (Heider et al., Mol Cell 2021). MCT1 expression was also specified as an independent negative prognostic marker for lenalidomide maintenance therapy within large MM patient cohorts, thus further underscoring the validity of this new mechanism of IMiD function (Stroh et al., Blood Adv 2022).
In addition to the above projects, various successful collaborations have been performed under the BCM-UPS funding and are specified in the “publications” section of this report.
First, our discovery of the molecular mode of action of IMiDs. While it was previously shown that the mediator of IMiD activity, CRBN, functions as the substrate-recruiting determinant of the CUL4-RBX1-DDB1 ubiquitin ligase complex (CRL4) that becomes re-directed toward neo-substrates such as IKZF1/3 by certain IMiDs, this mechanism created a paradoxon as IMiDs and proteasomal inhibitors are highly synergistic and suggested a ubiquitin-independent function of CRBN. Our studies found that CRBN exists in a CUL4 ligase independent state in which it mediates quality control of central transmembrane proteins such as MCT1/CD147 and CD98/LAT1. IMiDs outcompete the quality control function of CRBN on these proteins with the consequence of their misfolding and removal by the unfolded protein response, eventually leading to cell death of MM cells (Heider et al., Mol Cell 2021).
Our results on a mitotic transcriptional program via the USP9X deubiquitylase mark a truly unexpected finding. Mitosis has been considered transcriptionally silent. Our finding however demonstrate that distinct transcription factors (i.e. WT1) retain their activity in mitosis. Our data further demonstrate, that stabilization of WT1 via USP9X exerts an oncogenic transcriptional program by which WT1 promotes oncogenic IL8 signaling (Dietachmayr et al., Nat Communications 2020).
Our results on a mitotic transcriptional program via the USP9X deubiquitylase mark a truly unexpected finding. Mitosis has been considered transcriptionally silent. Our finding however demonstrate that distinct transcription factors (i.e. WT1) retain their activity in mitosis. Our data further demonstrate, that stabilization of WT1 via USP9X exerts an oncogenic transcriptional program by which WT1 promotes oncogenic IL8 signaling (Dietachmayr et al., Nat Communications 2020).