Current projects
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We deeply appreciate the support from organizations and individuals who make our work possible. Every contribution, big or small, directly funds research conducted in the Carrasco Lab. All donations are tax-deductible and help us advance our mission to improve the lives of patients with cancer.
We deeply appreciate the support from organizations and individuals who make our work possible. Every contribution, big or small, directly funds research conducted in the Carrasco Lab. All donations are tax-deductible and help us advance our mission to improve the lives of patients with cancer.
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Investigating and modeling MYD88L265P and co-occurring mutations in mature B-cell malignancies (R01CA273123)
Non-Hodgkin lymphomas (NHLs) of B-cell type, a heterogenous group of lymphoid malignancies, are among the most common cancers worldwide, accounting for about 4% of all cancers. A dramatic rise in incidence of NHLs worldwide during the past decades has sparked intense research efforts to understand their pathogenesis. Genomics studies have uncovered many novel genomic alterations in NHLs, but these remain to be functionally validated and characterized.
Among the most common of the genomic alterations is a missense mutation that results in leucine-to-proline substitution at position 265 in MYD88 (MYD88L265P), an adaptor protein that activates oncogenic NF-κB signaling. The MYD88L265P mutation is exceptionally frequent in lymphoplasmacytic lymphoma (LPL) and activated B-cell type of diffuse large B-cell lymphoma (ABC-DLBCL). While inhibition of MYD88L265P adversely impacts the survival of LPL and ABC-DLBCL cells, its role in lymphoma initiation remains to be clarified. Therefore, to elucidate the lymphomagenic potential of MYD88L265P we generated conditional transgenic mice overexpressing human wild-type (hMYD88WT) or mutant (hMYD88L265P) proteins in activated B-cells. Although abundance of both proteins and p65 NF-κB nuclear translocation was increased in transgenic GC B- cells, we observed that:
These results indicate that MYD88L265P possesses unique biochemical and functional properties, and suggest that the hMYD88WT and hMYD88L265P transgenic mice constitute an ideal model system in which to investigate these properties, as well as the secondary cooperating genetic alterations that are necessary to fully develop a clonal LPL phenotype and its eventual progression to ABC-DLBCL. We will investigate the role of the MYD88L265P mutation, Chr6q deletion (Chr10q in mice) as well as other LPL- associated loss-of-function gene mutations in B-cell development and function as well as the pathogenesis of LPL and ABC-DLBCL, and to develop a preclinical mouse models of LPL and ABC-DLBCL for testing therapies.
Non-Hodgkin lymphomas (NHLs) of B-cell type, a heterogenous group of lymphoid malignancies, are among the most common cancers worldwide, accounting for about 4% of all cancers. A dramatic rise in incidence of NHLs worldwide during the past decades has sparked intense research efforts to understand their pathogenesis. Genomics studies have uncovered many novel genomic alterations in NHLs, but these remain to be functionally validated and characterized.
Among the most common of the genomic alterations is a missense mutation that results in leucine-to-proline substitution at position 265 in MYD88 (MYD88L265P), an adaptor protein that activates oncogenic NF-κB signaling. The MYD88L265P mutation is exceptionally frequent in lymphoplasmacytic lymphoma (LPL) and activated B-cell type of diffuse large B-cell lymphoma (ABC-DLBCL). While inhibition of MYD88L265P adversely impacts the survival of LPL and ABC-DLBCL cells, its role in lymphoma initiation remains to be clarified. Therefore, to elucidate the lymphomagenic potential of MYD88L265P we generated conditional transgenic mice overexpressing human wild-type (hMYD88WT) or mutant (hMYD88L265P) proteins in activated B-cells. Although abundance of both proteins and p65 NF-κB nuclear translocation was increased in transgenic GC B- cells, we observed that:
- the MYD88L265P protein differed from the MYD88WT in its stability, ease of aggregation, and downstream activity;
- hMYD88WT did not produce detectable phenotypic alterations, but hMYD88L265P promoted with high frequency and long latency, a non-clonal, low-grade B-cell lymphoproliferative disorder resembling human LPL, which occasionally underwent transformation to ABC-DLBCL, suggesting that MYD88L265P is insufficient by itself to drive neoplastic transformation of mature B-cells, and that secondary cooperating genetic alterations are needed.
These results indicate that MYD88L265P possesses unique biochemical and functional properties, and suggest that the hMYD88WT and hMYD88L265P transgenic mice constitute an ideal model system in which to investigate these properties, as well as the secondary cooperating genetic alterations that are necessary to fully develop a clonal LPL phenotype and its eventual progression to ABC-DLBCL. We will investigate the role of the MYD88L265P mutation, Chr6q deletion (Chr10q in mice) as well as other LPL- associated loss-of-function gene mutations in B-cell development and function as well as the pathogenesis of LPL and ABC-DLBCL, and to develop a preclinical mouse models of LPL and ABC-DLBCL for testing therapies.
Mutated MYD88 signaling in Waldenstrom's Macroglobulinemia (Treon et al., Hemasphere, 2019)
Dissecting cancer cell-intrinsic and microenvironmental roles of MYD88 Mutations and Chromosome 6q deletions in Waldenstrom's Macroglobulinemia (Robert A. Kyle Career Development Award)
This project focuses on investigating the interplay between MYD88 mutations and chromosome 6q deletions in the development and progression of B-cell lymphomas, particularly Waldenström’s Macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL). Using genetically engineered mouse models, the study aims to unravel the molecular mechanisms driving lymphomagenesis, characterize tumor microenvironmental interactions, and identify novel therapeutic targets.
The research will leverage cutting-edge tools such as single-cell RNA sequencing, epigenetic profiling, and functional validation in both murine and human lymphoma models. Additionally, the project seeks to develop preclinical models for evaluating new therapeutic approaches, addressing unmet needs for WM and DLBCL patients by bridging basic research and clinical applications.
This project focuses on investigating the interplay between MYD88 mutations and chromosome 6q deletions in the development and progression of B-cell lymphomas, particularly Waldenström’s Macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL). Using genetically engineered mouse models, the study aims to unravel the molecular mechanisms driving lymphomagenesis, characterize tumor microenvironmental interactions, and identify novel therapeutic targets.
The research will leverage cutting-edge tools such as single-cell RNA sequencing, epigenetic profiling, and functional validation in both murine and human lymphoma models. Additionally, the project seeks to develop preclinical models for evaluating new therapeutic approaches, addressing unmet needs for WM and DLBCL patients by bridging basic research and clinical applications.
Development of microRNA (miR)-based cell-targeted polymeric nanoparticles for myeloma therapy (R01CA248393)
Multiple myeloma (MM), a cancer of plasma cells that colonize the bone marrow (BM), remains incurable despite the use of new promising treatment modalities. This is partly due to
We recently documented that:
The main challenge for miR-based therapy is the need for safe and effective delivery methods. Unless chemically modified or physically encapsulated, miRs are unstable in the blood and do not easily cross the cell membrane. Nanoparticles (NPs) encompass a variety of submicron-sized macromolecules that have been used successfully as vehicles for various agents, including miRs, enabling these agents to reach cellular targets previously considered undruggable. The Langer lab has successfully engineered a diverse library of polymeric NPs, of which one exemplar, 7C1NP, was shown to be non-toxic and effective in delivering siRNAs to BMECs in mice. My lab subsequently showed that the 7C1NP formulation can deliver siRNAs/miRs not only to human BMECs but also to MM cells as well as murine immune cells in vivo.
The overarching goal of this project is to take advantage of the 7C1NP delivery system to
The proposed studies are significant to public health in that they will be performed with MM cells lines and MM cells from patients and utilizing clinically relevant mouse xenograft models of MM that take in consideration the heterotypic interactions between MM cells and the BMME and their ultimate goal is to improve patient outcome with more efficacious therapies that alleviate suffering, and reduce the overall treatment cost of not only MM but potentially other hematologic malignancies.
Multiple myeloma (MM), a cancer of plasma cells that colonize the bone marrow (BM), remains incurable despite the use of new promising treatment modalities. This is partly due to
- MM progression and drug resistance development,
- protection of MM cells by the BM microenvironment (BMME),
- immune evasion.
We recently documented that:
- the miR-30-5p family serves as an MM-tumor suppressor targeting BCL9, a critical Wnt/β-catenin co-activator, highly expressed in BM endothelial cells (BMECs), that promotes BM colonization and proliferation of MM cells,
- the miR-221/222 cluster is overexpressed in MM cells from patients who have become unresponsive to dexamethasone, and functions as an MM oncogene by targeting PUMA and inhibiting apoptosis
- miR-30c-5p and miR-221/222 are expressed in murine immune cells, and we can identify murine macrophages within MM tumors engrafted in mice.
The main challenge for miR-based therapy is the need for safe and effective delivery methods. Unless chemically modified or physically encapsulated, miRs are unstable in the blood and do not easily cross the cell membrane. Nanoparticles (NPs) encompass a variety of submicron-sized macromolecules that have been used successfully as vehicles for various agents, including miRs, enabling these agents to reach cellular targets previously considered undruggable. The Langer lab has successfully engineered a diverse library of polymeric NPs, of which one exemplar, 7C1NP, was shown to be non-toxic and effective in delivering siRNAs to BMECs in mice. My lab subsequently showed that the 7C1NP formulation can deliver siRNAs/miRs not only to human BMECs but also to MM cells as well as murine immune cells in vivo.
The overarching goal of this project is to take advantage of the 7C1NP delivery system to
- uncover possible new targets of, and roles for, miR-30-5p and miR-221/222 in MM progression;
- explore the potential of these polymer-encapsulated miRs for MM therapy via
- miR-30-5p “replacement therapy” to target BCL9 in BMECs, and inhibit MM growth in the BM
- miR-221/222 “antisense (as) therapy” to target PUMA in MM cells and enhance apoptosis while abrogating acquired resistance to Lenalidomide, and Bortezomib
- investigate the effect of these therapies on other immune cells and MM-associated macrophage polarization.
The proposed studies are significant to public health in that they will be performed with MM cells lines and MM cells from patients and utilizing clinically relevant mouse xenograft models of MM that take in consideration the heterotypic interactions between MM cells and the BMME and their ultimate goal is to improve patient outcome with more efficacious therapies that alleviate suffering, and reduce the overall treatment cost of not only MM but potentially other hematologic malignancies.
Engineering lipid–polymer nanoparticles (NPs) for siRNA delivery to the bone marrow microenvironment for multiple myeloma therapy (Guimarães et al., PNAS, 2023)
Developing selective inhibitors of the β-catenin/BCL9 transcriptional complex for myeloma therapy (LLS Translational Research Program)
The β-catenin/BCL9 transcriptional complex, is a novel dependency in multiple myeloma (MM). Disruption of this complex inhibits MM cell growth in culture and in MM xenograft models. Development of potent selective β-catenin/BCL9 inhibitors will provide valuable tools to further investigate their mechanism of MM inhibition. We have established a chemistry, structural biology, and molecular pathology platform to facilitate novel inhibitor development, and explore its translational potential in MM.
Multiple myeloma (MM) is a cancer resulting from accumulation as multiple “omas” of malignant plasma cells in the bone marrow, the soft inner part of bones where new blood cells formation takes place. MM is the second most frequent human blood cancer, which remains incurable despite the advent of promising approaches such as CAR T-cell therapy. This is due, in part, to disease heterogeneity and clonal evolution underlying development of drug resistance and disease progression. Thus, there remains an urgent unmet medical need for innovative therapies, particularly for patients with advanced disease unresponsive to available therapies.
The Wnt/β-catenin system is a signaling pathway used by the tumor cells to communicate each other and with the tumor microenvironment. It conveys extracellular information from the cell surface to the nucleus where it enhances expression of genes involved in sustaining cancer stemness and promoting epithelial-to-mesenchymal transition and tumor immune evasion. Inappropriate activation of the Wnt/β-catenin pathway contributes to the initiation and progression of various human cancer types, including MM, and has emerged as a desirable, though challenging therapeutic target.
BCL9 is a critical co-activator of the Wnt/β-catenin pathway via direct binding to β-catenin. Data from our lab suggest that targeted disruption of the β-catenin/BCL9 heterodimeric complex is a possible therapeutic approach for MM based on the following findings:
The finding that statin use is associated with reduced mortality among MM patients underscore the relevance of our findings and highlights the need for developing more effective C-1 derivatives and study their role in oncogenic Wnt/β-catenin activity, cholesterol metabolism and MM biology and therapy. With that purpose, we have inaugurated a close partnership that will bring together researchers with complementary expertise in structural biology (Dhe-Paganon lab), medicinal chemistry (Qi Lab), molecular pathology (Carrasco Lab), and clinical medicine (Anderson Lab) to facilitate novel inhibitor development, and explore its translational potential in MM.
The β-catenin/BCL9 transcriptional complex, is a novel dependency in multiple myeloma (MM). Disruption of this complex inhibits MM cell growth in culture and in MM xenograft models. Development of potent selective β-catenin/BCL9 inhibitors will provide valuable tools to further investigate their mechanism of MM inhibition. We have established a chemistry, structural biology, and molecular pathology platform to facilitate novel inhibitor development, and explore its translational potential in MM.
Multiple myeloma (MM) is a cancer resulting from accumulation as multiple “omas” of malignant plasma cells in the bone marrow, the soft inner part of bones where new blood cells formation takes place. MM is the second most frequent human blood cancer, which remains incurable despite the advent of promising approaches such as CAR T-cell therapy. This is due, in part, to disease heterogeneity and clonal evolution underlying development of drug resistance and disease progression. Thus, there remains an urgent unmet medical need for innovative therapies, particularly for patients with advanced disease unresponsive to available therapies.
The Wnt/β-catenin system is a signaling pathway used by the tumor cells to communicate each other and with the tumor microenvironment. It conveys extracellular information from the cell surface to the nucleus where it enhances expression of genes involved in sustaining cancer stemness and promoting epithelial-to-mesenchymal transition and tumor immune evasion. Inappropriate activation of the Wnt/β-catenin pathway contributes to the initiation and progression of various human cancer types, including MM, and has emerged as a desirable, though challenging therapeutic target.
BCL9 is a critical co-activator of the Wnt/β-catenin pathway via direct binding to β-catenin. Data from our lab suggest that targeted disruption of the β-catenin/BCL9 heterodimeric complex is a possible therapeutic approach for MM based on the following findings:
- BCL9 is frequently over-expressed in MM cells but is absent in the normal plasma cells, providing a broad therapeutic window for targeting this complex;
- The β-catenin/BCL9 complex serves multiple roles in MM pathogenesis and disease progression;
- Deployment of a robust high-throughput screen assay with a small-molecule compound library allowed us to identify C-1 a “lead compound” that specifically disrupted formation of the β-catenin/BCL9 complex. C-1 treatment is associated with inhibition of cholesterol metabolism and anti-tumor activity in culture cells and in a murine model of MM. C-1 reduces MM tumor growth alone, but further reduces tumor burden when given in combination with other cholesterol lowering drugs or statins.
The finding that statin use is associated with reduced mortality among MM patients underscore the relevance of our findings and highlights the need for developing more effective C-1 derivatives and study their role in oncogenic Wnt/β-catenin activity, cholesterol metabolism and MM biology and therapy. With that purpose, we have inaugurated a close partnership that will bring together researchers with complementary expertise in structural biology (Dhe-Paganon lab), medicinal chemistry (Qi Lab), molecular pathology (Carrasco Lab), and clinical medicine (Anderson Lab) to facilitate novel inhibitor development, and explore its translational potential in MM.
Model of C-1 mechanism of action (Tanton, Sewastianik et al., Science Advances, 2022)
Pathology (P01CA206978)
This project focuses on developing a robust infrastructure to support research on Richter Syndrome (RS) by utilizing patient-derived samples and advanced modeling techniques. A key component is the ex vivo expansion of primary RS samples in immunocompromised mice to create in vivo primagraft models. These models will provide fresh or frozen RS cells for both in vitro molecular analyses and in vivo functional experiments, enabling detailed studies of RS biology, signaling pathways, genetic alterations, and mechanisms of drug resistance.
RS samples will be sourced from an existing biorepository and newly enrolled patients, accompanied by full clinical and molecular annotation. These models will be essential for validating therapeutic targets and assessing drug responses. Primary RS cells will be delivered via tail vein injection or implanted subcutaneously or beneath the renal capsule to facilitate tumor growth and characterization.
In addition to generating and maintaining these models, the project will:
This project focuses on developing a robust infrastructure to support research on Richter Syndrome (RS) by utilizing patient-derived samples and advanced modeling techniques. A key component is the ex vivo expansion of primary RS samples in immunocompromised mice to create in vivo primagraft models. These models will provide fresh or frozen RS cells for both in vitro molecular analyses and in vivo functional experiments, enabling detailed studies of RS biology, signaling pathways, genetic alterations, and mechanisms of drug resistance.
RS samples will be sourced from an existing biorepository and newly enrolled patients, accompanied by full clinical and molecular annotation. These models will be essential for validating therapeutic targets and assessing drug responses. Primary RS cells will be delivered via tail vein injection or implanted subcutaneously or beneath the renal capsule to facilitate tumor growth and characterization.
In addition to generating and maintaining these models, the project will:
- Provide formalin-fixed, paraffin-embedded (FFPE) RS tissue samples for histopathological studies.
- Conduct histopathological analyses of human RS and mouse-engineered RS samples.
- Create tissue microarrays and matched CLL/RS sample slides for validation studies.
- Perform immunohistochemical and morphological characterizations of RS tissues in engineered mouse models.
In addition to the projects mentioned, we are also involved in several exploratory projects that remain confidential at this stage. If you're interested in applying for a position or establishing a collaboration, we’d be happy to discuss the nature of these projects further.
Past projects
MYD88L265P signaling-associated multiplex characterization of the bone marrow microenvironment in WM patients for clinical application
Thanks to the support of the International Waldenström's Macroglobulinemia Foundation (IWMF) and the Leukemia & Lymphoma Society (LLS), we recently generated conditional transgenic mice overexpressing MYD88WT or MYD88L265P human proteins in activated B-cells, and found that MYD88L265P leads to (I) formation of protein aggregates resembling myddosome/My-T-BCR in plasmacytoid lymphocytes and (II) significantly higher T-cell and mast cell numbers in the BM of both our murine models as well as in a validation set of WM patients’ biopsies. The presence of focal MYD88L265P protein aggregates was recently shown to be a functional and predictive factor in DLBCL, while changes in specific immune and stromal cell components of the BM microenvironment regulate tumor cell proliferation and survival, and are correlated with disease progression, drug resistance, and overall outcome in WM just as they are in other hematologic malignancies. Thus, our overarching goals for this project are to translate and expand the findings from our mouse models with the goal to (I) confirm MYD88L265P aggregates as a clinically relevant diagnostic assay (and ultimately a validated marker) and use it to study MYD88L265P signaling at a single-cell level, and (II) systematically characterize the cellular components of the BM microenvironment during WM disease progression, investigate its functional role, and identify potential clinically exploitable vulnerabilities.
Thanks to the support of the International Waldenström's Macroglobulinemia Foundation (IWMF) and the Leukemia & Lymphoma Society (LLS), we recently generated conditional transgenic mice overexpressing MYD88WT or MYD88L265P human proteins in activated B-cells, and found that MYD88L265P leads to (I) formation of protein aggregates resembling myddosome/My-T-BCR in plasmacytoid lymphocytes and (II) significantly higher T-cell and mast cell numbers in the BM of both our murine models as well as in a validation set of WM patients’ biopsies. The presence of focal MYD88L265P protein aggregates was recently shown to be a functional and predictive factor in DLBCL, while changes in specific immune and stromal cell components of the BM microenvironment regulate tumor cell proliferation and survival, and are correlated with disease progression, drug resistance, and overall outcome in WM just as they are in other hematologic malignancies. Thus, our overarching goals for this project are to translate and expand the findings from our mouse models with the goal to (I) confirm MYD88L265P aggregates as a clinically relevant diagnostic assay (and ultimately a validated marker) and use it to study MYD88L265P signaling at a single-cell level, and (II) systematically characterize the cellular components of the BM microenvironment during WM disease progression, investigate its functional role, and identify potential clinically exploitable vulnerabilities.
Mining B-catenin/BCL9 transcriptional complex for Multiple Myeloma therapeutics (R01CA151391)
Multiple Myeloma (MM) is the second most frequent hematological cancer in the US after non- Hodgkin's lymphoma with about 20,000 individuals succumbing to this dreaded disease each year in the US alone. Despite recent advances in its treatment, the median survival remains at 6 years, with only 10% of patients surviving at 10 years. Therefore there is an urgent need for new and effective therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, and shared across different MM subtypes. Our laboratory has devoted significant effort towards the identification of the molecular genetic events in this malignancy, with the goals of improving early detection and providing new molecular targets for the development of more effective therapies for this cancer. Preliminary data: In our previous studies we have documented that the Wnt/B-catenin/BCL9 pathway is one of such pathway involved in MM disease progression and maintenance. Specifically we have found that: i) BCL9 is overexpressed in most MM cells but it is not expressed in the normal cellular counterpart where they originate, ii) BCL9 promotes tumor progression by conferring enhanced proliferative, metastatic and angiogenic properties to myeloma cells, iii) RNAi suppressed expression of either 2-catenin or BCL9 inhibits MM tumor growth in vitro and in vivo, and iv) stapled peptides of the BCL9 HD2 domain inhibit 2-catenin/BCL9-dependent transcriptional activity in MM cells. Working hypothesis: The 2-catenin/BCL9 transcriptional complex itself and some of the downstream transcriptional targets are novel important therapeutic targets in MM. Goals: Our goals are to validate and functionally characterize novel B-catenin/BCL9 transcriptional target genes and to explore the possible role of the 2-catenin/BCL9 protein complex itself as therapeutic target in MM. Experimental tools: To test our hypothesis we will use as tools stabilized alpha-helices of BCL9 to disrupt B-catenin/BCL9 protein interaction and stabilized nano particles to deliver BCL9 small interfering RNAs to myeloma cells. In addition, we will use our expertise with lentiviral-based gene transfer technologies for functional validation, using gain- and loss-of-function approaches as well as well-established in vitro and in vivo model systems that reflect the heterotopic interactions between the MM cell and bone marrow microenvironment. Expected results: i) to identify and validate novel downstream B-catenin/BCL9 downstream genes which could be used as therapeutic targets in MM, ii) to functionally characterize and validate the role of the B-catenin/BCL9 transcriptional complex itself as a novel therapeutic target in MM. Probable implications to Medicine: The potential implications are: i) to find novel genes involved in the pathogenesis and progression of MM, ii) to find novel molecular targets to effectively treat MM, and iii) to develop preclinical models for designing and assessing target-based therapeutic approaches in MM and other hematologic malignancies associated with dysregulated Wnt activity.
Multiple Myeloma (MM) is the second most frequent hematological cancer in the US after non- Hodgkin's lymphoma with about 20,000 individuals succumbing to this dreaded disease each year in the US alone. Despite recent advances in its treatment, the median survival remains at 6 years, with only 10% of patients surviving at 10 years. Therefore there is an urgent need for new and effective therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, and shared across different MM subtypes. Our laboratory has devoted significant effort towards the identification of the molecular genetic events in this malignancy, with the goals of improving early detection and providing new molecular targets for the development of more effective therapies for this cancer. Preliminary data: In our previous studies we have documented that the Wnt/B-catenin/BCL9 pathway is one of such pathway involved in MM disease progression and maintenance. Specifically we have found that: i) BCL9 is overexpressed in most MM cells but it is not expressed in the normal cellular counterpart where they originate, ii) BCL9 promotes tumor progression by conferring enhanced proliferative, metastatic and angiogenic properties to myeloma cells, iii) RNAi suppressed expression of either 2-catenin or BCL9 inhibits MM tumor growth in vitro and in vivo, and iv) stapled peptides of the BCL9 HD2 domain inhibit 2-catenin/BCL9-dependent transcriptional activity in MM cells. Working hypothesis: The 2-catenin/BCL9 transcriptional complex itself and some of the downstream transcriptional targets are novel important therapeutic targets in MM. Goals: Our goals are to validate and functionally characterize novel B-catenin/BCL9 transcriptional target genes and to explore the possible role of the 2-catenin/BCL9 protein complex itself as therapeutic target in MM. Experimental tools: To test our hypothesis we will use as tools stabilized alpha-helices of BCL9 to disrupt B-catenin/BCL9 protein interaction and stabilized nano particles to deliver BCL9 small interfering RNAs to myeloma cells. In addition, we will use our expertise with lentiviral-based gene transfer technologies for functional validation, using gain- and loss-of-function approaches as well as well-established in vitro and in vivo model systems that reflect the heterotopic interactions between the MM cell and bone marrow microenvironment. Expected results: i) to identify and validate novel downstream B-catenin/BCL9 downstream genes which could be used as therapeutic targets in MM, ii) to functionally characterize and validate the role of the B-catenin/BCL9 transcriptional complex itself as a novel therapeutic target in MM. Probable implications to Medicine: The potential implications are: i) to find novel genes involved in the pathogenesis and progression of MM, ii) to find novel molecular targets to effectively treat MM, and iii) to develop preclinical models for designing and assessing target-based therapeutic approaches in MM and other hematologic malignancies associated with dysregulated Wnt activity.
Development of molecular probes to investigate and drug the oncogenic Wnt/β-catenin/BCL9/B9L transcriptional complex (R21CA221683)
Dysregulated Wnt/β-catenin signaling has been implicated in the pathogenesis of many common human cancers, making this an attractive clinical target. However, this has proven challenging because i) β- catenin plays critical roles in normal tissue homeostasis, ii) its signaling pathways form part of a complex network of intersecting pathways, and iii) β-catenin's mode of interaction with active sites on its partner proteins makes it difficult to identify molecular probes that specifically and selectively disrupt its oncogenic activity. Preliminary data: i) Development of peptidomimetics of the BCL9-HD2 domain that selectively suppress oncogenic Wnt activity by blocking interaction of β-catenin with its transcriptional co-activator BCL9; ii) A high-throughput screening (HTS) AlphaScreen assay allowing identification of several small-molecule inhibitors of β-catenin/BCL9 interaction has been implemented; iii) An in vivo model using BCL9 transgenic mice that develop lymphomas as well as lung and gastric adenocarcinomas has been put into place. These studies offer compelling proof-of-concept for i) a therapeutic strategy addressing a known oncogenic role of the Wnt/β-catenin/BCL9 transcriptional complex, and ii) a pharmacologic intervention via targeted disruption of this complex. Working hypothesis: i) The Wnt/β-catenin/BCL9 transcriptional complex performs critical roles in cancer pathogenesis, and targeted disruption of this complex represents a promising pharmacologic strategy for blocking oncogenic Wnt activity in cancer; ii) Potent, specific, competitive inhibition of the β-catenin/BCL9 interaction can be accomplished by using small organic molecules that bind tightly to the BCL9-HD2 binding domain on β-catenin. Goals: i) to characterize HTS “hits” biophysically via rapid calorimetry-based assay; ii) to test inhibitors for functional activity in Wnt-relevant cell-based assays to ensure that the inhibitors have the desired molecular specificity. Experimental tools: i) Homogeneous biophysical assays (i.e. enthalpy change measurement and Isothermal titration calorimetry) that will robustly monitor disruption of β-catenin/BCL9 binding in vitro, ii) X-ray crystallography to unequivocally establish the structural basis of this interaction and will lay groundwork for possible future structure-guided synthetic chemistry; iii) Cellular assays that will enable us to monitor the consequences of disrupting the β-catenin/BCL9 complex in intact cells; and iv) Established xenograft and transgenic mouse models of cancers with a dysregulated Wnt/β-catenin/BCL9 complex will allow evaluation of the possible clinical usefulness of small-molecule inhibitors of BCL9/β-catenin interaction. Expected results: Structurally validated small-molecule chemical probes that i) target β-catenin, ii) dissociate native β-catenin/BCL9 complexes, iii) selectively suppress Wnt transcriptional activity, and iv) possess mechanism-based antitumor activity in vitro, and in vivo with minimal toxicity, will be identified. Implications to Medicine: Our work in this project is expected to afford highly selective clinical probes of oncogenic Wnt activity and innovative targeted therapies against Wnt/β-catenin/BCL9 dependent human cancers.
Dysregulated Wnt/β-catenin signaling has been implicated in the pathogenesis of many common human cancers, making this an attractive clinical target. However, this has proven challenging because i) β- catenin plays critical roles in normal tissue homeostasis, ii) its signaling pathways form part of a complex network of intersecting pathways, and iii) β-catenin's mode of interaction with active sites on its partner proteins makes it difficult to identify molecular probes that specifically and selectively disrupt its oncogenic activity. Preliminary data: i) Development of peptidomimetics of the BCL9-HD2 domain that selectively suppress oncogenic Wnt activity by blocking interaction of β-catenin with its transcriptional co-activator BCL9; ii) A high-throughput screening (HTS) AlphaScreen assay allowing identification of several small-molecule inhibitors of β-catenin/BCL9 interaction has been implemented; iii) An in vivo model using BCL9 transgenic mice that develop lymphomas as well as lung and gastric adenocarcinomas has been put into place. These studies offer compelling proof-of-concept for i) a therapeutic strategy addressing a known oncogenic role of the Wnt/β-catenin/BCL9 transcriptional complex, and ii) a pharmacologic intervention via targeted disruption of this complex. Working hypothesis: i) The Wnt/β-catenin/BCL9 transcriptional complex performs critical roles in cancer pathogenesis, and targeted disruption of this complex represents a promising pharmacologic strategy for blocking oncogenic Wnt activity in cancer; ii) Potent, specific, competitive inhibition of the β-catenin/BCL9 interaction can be accomplished by using small organic molecules that bind tightly to the BCL9-HD2 binding domain on β-catenin. Goals: i) to characterize HTS “hits” biophysically via rapid calorimetry-based assay; ii) to test inhibitors for functional activity in Wnt-relevant cell-based assays to ensure that the inhibitors have the desired molecular specificity. Experimental tools: i) Homogeneous biophysical assays (i.e. enthalpy change measurement and Isothermal titration calorimetry) that will robustly monitor disruption of β-catenin/BCL9 binding in vitro, ii) X-ray crystallography to unequivocally establish the structural basis of this interaction and will lay groundwork for possible future structure-guided synthetic chemistry; iii) Cellular assays that will enable us to monitor the consequences of disrupting the β-catenin/BCL9 complex in intact cells; and iv) Established xenograft and transgenic mouse models of cancers with a dysregulated Wnt/β-catenin/BCL9 complex will allow evaluation of the possible clinical usefulness of small-molecule inhibitors of BCL9/β-catenin interaction. Expected results: Structurally validated small-molecule chemical probes that i) target β-catenin, ii) dissociate native β-catenin/BCL9 complexes, iii) selectively suppress Wnt transcriptional activity, and iv) possess mechanism-based antitumor activity in vitro, and in vivo with minimal toxicity, will be identified. Implications to Medicine: Our work in this project is expected to afford highly selective clinical probes of oncogenic Wnt activity and innovative targeted therapies against Wnt/β-catenin/BCL9 dependent human cancers.
Validating the eCyPA/CD147 signaling complex for myeloma therapy (R01CA196783)
Multiple Myeloma (MM) is a cancer of plasma cells that accumulate in the bone marrow (BM). Despite recent advances in treatments, it remains incurable and there is urgent need of novel and more effective therapies. However, there has recently surfaced a new treatment paradigm that shows great promise to improve patient outcome by disrupting the tropism that the BM microenvironment, the `soil', plays on MM cells, the `seeds'. Since BM angiogenesis is a hallmark of MM progression that correlates with disease stage, it became evident that among the interactions between MM cells and the BM microenvironment, those with BM endothelial cells (BMECs) must play an important role in MM progression. Preliminary data: During studies of the interaction of MM cells with the BM microenvironment, we uncovered a critical role of canonical Wnt signaling, a conduit for cell-cell communication and tropism culminating in transcriptional activation of pro- migration, proliferation, and survival genes. The terminal effector of Wnt signaling is a transcriptional complex that includes two other signaling proteins, ß-catenin and BCL9. Moreover, during immunohistochemical studies using BM tissue microarrays, we observed restricted and high-level BCL9 expression in BMECs but not other BM cells. In addition, using proteomic analysis we have documented that extracellular Cyclophilin A (eCyPA) is a downstream transcriptional target of the Wnt/ß-catenin/BCL9 complex, which is secreted by BMECs but not other BM stromal cells and promote pleiotropic signaling changes in MM including enhanced expression of CD14, the know receptor of eCyPA. Furthermore, knockdown of either eCyPA in BMECs or CD147 in MM cells markedly decreased migration and proliferation of MM cells. Working hypothesis: (i) signaling from BMECs is essential for MM progression; (ii) eCyPA plays critical roles in the signaling output from BMECs that modulate migration, invasion, colonization, growth, survival, and drug resistance of MM cells. Thus, targeting the interaction between eCyPA and its cognate receptor CD147 on MM cells is therapeutic for MM, particularly for cases with acquired resistance to standard chemotherapy. Goals: (i) to further characterize the role of BMECs in MM progression, (ii) to validate the role of the eCyPA/CD147 signaling complex as therapeutic target for MM, (iii) to identify and functionally characterize additional signaling molecules secreted by BMECs that promote MM progression, and (iii) to develop a high throughput screening assay to identify small molecule inhibitors of eCyPA/CD147 interaction for future development of targeted therapies for MM. Expected results: i) validate role of eCyPA/CD147 signaling complex as effective nontoxic target for MM therapy; ii) identification of novel potential biomarkers of MM progression and therapeutic targets. Broader implications for medicine: Development of more effective targeted therapies for MM and other hematologic malignancies that express CD147 and 'home in' the BM.
Multiple Myeloma (MM) is a cancer of plasma cells that accumulate in the bone marrow (BM). Despite recent advances in treatments, it remains incurable and there is urgent need of novel and more effective therapies. However, there has recently surfaced a new treatment paradigm that shows great promise to improve patient outcome by disrupting the tropism that the BM microenvironment, the `soil', plays on MM cells, the `seeds'. Since BM angiogenesis is a hallmark of MM progression that correlates with disease stage, it became evident that among the interactions between MM cells and the BM microenvironment, those with BM endothelial cells (BMECs) must play an important role in MM progression. Preliminary data: During studies of the interaction of MM cells with the BM microenvironment, we uncovered a critical role of canonical Wnt signaling, a conduit for cell-cell communication and tropism culminating in transcriptional activation of pro- migration, proliferation, and survival genes. The terminal effector of Wnt signaling is a transcriptional complex that includes two other signaling proteins, ß-catenin and BCL9. Moreover, during immunohistochemical studies using BM tissue microarrays, we observed restricted and high-level BCL9 expression in BMECs but not other BM cells. In addition, using proteomic analysis we have documented that extracellular Cyclophilin A (eCyPA) is a downstream transcriptional target of the Wnt/ß-catenin/BCL9 complex, which is secreted by BMECs but not other BM stromal cells and promote pleiotropic signaling changes in MM including enhanced expression of CD14, the know receptor of eCyPA. Furthermore, knockdown of either eCyPA in BMECs or CD147 in MM cells markedly decreased migration and proliferation of MM cells. Working hypothesis: (i) signaling from BMECs is essential for MM progression; (ii) eCyPA plays critical roles in the signaling output from BMECs that modulate migration, invasion, colonization, growth, survival, and drug resistance of MM cells. Thus, targeting the interaction between eCyPA and its cognate receptor CD147 on MM cells is therapeutic for MM, particularly for cases with acquired resistance to standard chemotherapy. Goals: (i) to further characterize the role of BMECs in MM progression, (ii) to validate the role of the eCyPA/CD147 signaling complex as therapeutic target for MM, (iii) to identify and functionally characterize additional signaling molecules secreted by BMECs that promote MM progression, and (iii) to develop a high throughput screening assay to identify small molecule inhibitors of eCyPA/CD147 interaction for future development of targeted therapies for MM. Expected results: i) validate role of eCyPA/CD147 signaling complex as effective nontoxic target for MM therapy; ii) identification of novel potential biomarkers of MM progression and therapeutic targets. Broader implications for medicine: Development of more effective targeted therapies for MM and other hematologic malignancies that express CD147 and 'home in' the BM.
Translating cancer genomics into therapeutic interventions in multiple myeloma
The main focus of the Carrasco Lab’s research is to investigate the role of the Wnt/β-catenin/BCL9 transcriptional complex in the pathogenesis of MM. Wnt signaling underlies the pathogenesis of a broad range of human cancers including carcinomas and hematological malignancies such as MM, yet the development of targeted therapies to disrupt the pathway has remained a formidable challenge due to (i) the toxicities associated with disrupting the pathway’s homeostatic functions and (ii) the large protein interaction surfaces involved. BCL9 is a co-activator of β-catenin-mediated transcription and is highly expressed in tumors but not in their cells of origin, presenting an opportunity to selectively block oncogenic Wnt activity. Having observed in our previous studies that BCL9 is frequently amplified and over-expressed in MM, we began to focus intensively on the role of this Wnt/β-catenin transcriptional co-activator in MM pathogenesis. Our team was the first to show that BCL9 functions as an oncogene that promotes MM progression by conferring enhanced proliferative, metastatic and angiogenic properties to myeloma cells. We next showed that Cyclophilin A (CyPA) is a downstream transcriptional target of the Wnt/β-catenin/BCL9 complex that is secreted by endothelial, but not other, bone marrow cells, and that this secreted factor (eCyPA) promotes pleiotropic signaling changes in MM, including enhanced expression of CD147, the known surface receptor of eCyPA, thereby enhancing the migration, proliferation, and bone marrow homing of MM cells. Having shown that BCL9 drives β-catenin signaling through a direct binding interaction mediated by its α-helical homology domain-2, we found in collaboration with Dr. Loren Walensky at the DFCI that a small “stapled” peptide called Stabilized Alpha-Helix of BCL9 (SAH-BCL9) targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and elicits mechanism-based antitumor responses. The clinical translation potential of this approach was underscored by the ability of SAH-BCL9 to effectively suppress the growth, angiogenesis, invasion, and metastasis of MM in vivo. Overall these studies highlight an important role of the Wnt/β-catenin/BCL9 transcriptional complex in MM disease progression and bone marrow homing, and provide compelling proof-of-concept for an innovative pharmacologic strategy to inhibit oncogenic Wnt signaling in MM as well as other cancers with dysregulated Wnt activity via targeted disruption of BCL9/β-catenin complex.
The main focus of the Carrasco Lab’s research is to investigate the role of the Wnt/β-catenin/BCL9 transcriptional complex in the pathogenesis of MM. Wnt signaling underlies the pathogenesis of a broad range of human cancers including carcinomas and hematological malignancies such as MM, yet the development of targeted therapies to disrupt the pathway has remained a formidable challenge due to (i) the toxicities associated with disrupting the pathway’s homeostatic functions and (ii) the large protein interaction surfaces involved. BCL9 is a co-activator of β-catenin-mediated transcription and is highly expressed in tumors but not in their cells of origin, presenting an opportunity to selectively block oncogenic Wnt activity. Having observed in our previous studies that BCL9 is frequently amplified and over-expressed in MM, we began to focus intensively on the role of this Wnt/β-catenin transcriptional co-activator in MM pathogenesis. Our team was the first to show that BCL9 functions as an oncogene that promotes MM progression by conferring enhanced proliferative, metastatic and angiogenic properties to myeloma cells. We next showed that Cyclophilin A (CyPA) is a downstream transcriptional target of the Wnt/β-catenin/BCL9 complex that is secreted by endothelial, but not other, bone marrow cells, and that this secreted factor (eCyPA) promotes pleiotropic signaling changes in MM, including enhanced expression of CD147, the known surface receptor of eCyPA, thereby enhancing the migration, proliferation, and bone marrow homing of MM cells. Having shown that BCL9 drives β-catenin signaling through a direct binding interaction mediated by its α-helical homology domain-2, we found in collaboration with Dr. Loren Walensky at the DFCI that a small “stapled” peptide called Stabilized Alpha-Helix of BCL9 (SAH-BCL9) targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and elicits mechanism-based antitumor responses. The clinical translation potential of this approach was underscored by the ability of SAH-BCL9 to effectively suppress the growth, angiogenesis, invasion, and metastasis of MM in vivo. Overall these studies highlight an important role of the Wnt/β-catenin/BCL9 transcriptional complex in MM disease progression and bone marrow homing, and provide compelling proof-of-concept for an innovative pharmacologic strategy to inhibit oncogenic Wnt signaling in MM as well as other cancers with dysregulated Wnt activity via targeted disruption of BCL9/β-catenin complex.
Defining the genomic landscape and biology of multiple myeloma
During the beginning of his time at the Dana-Farber Cancer Institute, Dr. Carrasco worked in the laboratory of Dr. Ronald DePinho (now at the M.D. Anderson Cancer Center in Houston). He directed his research efforts toward oncogenomics and the genetic mouse modeling of hematologic cancers and generated the first "bona fide" mouse model of histiocytic sarcoma, a highly malignant neoplasm of tissue macrophages using PTEN/INK/ARF conditional knockout mice. Subsequently his studies evolved to focus on multiple myeloma (MM), a malignancy of clonal plasma cells that colonize the bone marrow. Specifically, he used genome-wide array-comparative genomic hybridization (a-CGH) to characterize the multiple MM genomes, and generated a novel murine transgenic model of MM using the X-box binding protein 1 (XBP-1). These studies provided new insights into the roles of PTEN and INK-ARF tumor suppressor genes in mouse hematolymphoid development and XBP-1 gene in plasma cell development and pathogenesis of MM. In addition, these studies provided a comprehensive and integrated view of genes that are defective in human MM.
During the beginning of his time at the Dana-Farber Cancer Institute, Dr. Carrasco worked in the laboratory of Dr. Ronald DePinho (now at the M.D. Anderson Cancer Center in Houston). He directed his research efforts toward oncogenomics and the genetic mouse modeling of hematologic cancers and generated the first "bona fide" mouse model of histiocytic sarcoma, a highly malignant neoplasm of tissue macrophages using PTEN/INK/ARF conditional knockout mice. Subsequently his studies evolved to focus on multiple myeloma (MM), a malignancy of clonal plasma cells that colonize the bone marrow. Specifically, he used genome-wide array-comparative genomic hybridization (a-CGH) to characterize the multiple MM genomes, and generated a novel murine transgenic model of MM using the X-box binding protein 1 (XBP-1). These studies provided new insights into the roles of PTEN and INK-ARF tumor suppressor genes in mouse hematolymphoid development and XBP-1 gene in plasma cell development and pathogenesis of MM. In addition, these studies provided a comprehensive and integrated view of genes that are defective in human MM.
Anti-mR-221–222 therapy, by increasing the level of PUMA, which is downstream of GR and p53, may abrogate drug resistance associated with p53 inactivation and decreased GR expression (Zhao et al., Cancer Research, 2015)
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Defining the role of miRs in the pathogenesis of multiple myeloma
A secondary focus of our laboratory is to investigate the role of miRs in the pathogenesis of MM. MiRs are small (~22nt) noncoding RNAs that negatively regulate protein-coding gene expression by targeting mRNA degradation or translation inhibition. Dysregulation of miR expression is frequently observed in human cancers including MM, and has been associated with progressive disease, metastasis, drug resistance, and poor clinical outcome suggesting an important role of miRs in tumor progression. MiRs can function as either tumor suppressors or oncogenes, and have aroused interest as potential developmental foci for cancer therapy. In our recent studies we have demonstrated that the miR-30-5p family is frequently downregulated in MM and functions as a tumor suppressor and novel therapeutic tool by targeting oncogenic Wnt/β-catenin/BCL9 pathway, in addition we have found that the miR-221-222 family is frequently overexpressed in MM and anti-miR-221-222 therapy abrogates dexamethasone resistance by targeting the PUMA/BAK/BAX pathway. These studies point for the first time to a key role of the miR-30-5p and miR-221-222 families in the pathogenesis of MM, and provide compelling proof-of-concept for the potential exploitation of their role in MM therapy. |
Novel System to Study Telomere Dynamics in Hemotopoiesis (K08AG001031)
The termini of most eukaryotic chromosomes are composed of terminal repeats called telomeres. These repeats serve to protect chromosome integrity and ensure complete replication of essential genes. Although the molecular basis of aging or replicative senescence is not fully understood, reductions in telomere length are thought to play an important role. For example, telomeres are shorter in somatic tissues from older than from younger individuals. Children born with Hutchinson-Gilford progeria, an early-aging syndrome, have shorter telomeres than do age-matched controls. In other aging-genetics disorders, such as Werner's syndrome and Down's syndrome, cells lose telomeres at two to three times the rate in age-matched controls. Telomeric repeats are produced de novo by the ribonucleoprotein enzyme telomerase. The ectopic expression of telomerase in normal human somatic cells results in an extended lifespan and inhibition of replicative senescence. Telomeres also shorten in hematopoietic stem cells during normal aging and in the hematopoietic cells of young patients who have received allogenic bone marrow transplant. We postulate that if we can experimentally elongate the telomere ex vivo in normal human hematopoietic cells, replicative capacity and self-renewal potential may increase and thus alter the incidence and/or time of onset of age related hematopoietic disorders and immunosenescence. We propose to develop a mouse model system that permits the study of telomerase function in human hematopoietic cells. To this end, we will make use of the NOD/SCID mouse model in which the hematopoietic compartment can be reconstituted with human hematopoietic stem cells. Retroviral methods will be used to direct altered telomerase activity in these transplanted hematopoietic stem cells and their mature derivatives.
The termini of most eukaryotic chromosomes are composed of terminal repeats called telomeres. These repeats serve to protect chromosome integrity and ensure complete replication of essential genes. Although the molecular basis of aging or replicative senescence is not fully understood, reductions in telomere length are thought to play an important role. For example, telomeres are shorter in somatic tissues from older than from younger individuals. Children born with Hutchinson-Gilford progeria, an early-aging syndrome, have shorter telomeres than do age-matched controls. In other aging-genetics disorders, such as Werner's syndrome and Down's syndrome, cells lose telomeres at two to three times the rate in age-matched controls. Telomeric repeats are produced de novo by the ribonucleoprotein enzyme telomerase. The ectopic expression of telomerase in normal human somatic cells results in an extended lifespan and inhibition of replicative senescence. Telomeres also shorten in hematopoietic stem cells during normal aging and in the hematopoietic cells of young patients who have received allogenic bone marrow transplant. We postulate that if we can experimentally elongate the telomere ex vivo in normal human hematopoietic cells, replicative capacity and self-renewal potential may increase and thus alter the incidence and/or time of onset of age related hematopoietic disorders and immunosenescence. We propose to develop a mouse model system that permits the study of telomerase function in human hematopoietic cells. To this end, we will make use of the NOD/SCID mouse model in which the hematopoietic compartment can be reconstituted with human hematopoietic stem cells. Retroviral methods will be used to direct altered telomerase activity in these transplanted hematopoietic stem cells and their mature derivatives.