CRISPR-BASED FUNCTIONAL CHARACTERIZATION OF WM CELLS: INSIGHTS INTO THERAPEUTIC VULNERABILITIES AND STRATEGIES TO OVERCOME RESISTANCE

This project falls under the IWMF-LLS Strategic Research Roadmap Initiative. The research takes advantage of new technologies, including the gene editing tool CRISPR, improved and powerful computational approaches, and innovative new mouse models. The researchers will conduct a broad, genome-wide search to identify specific genes that are required to allow Waldenström’s macroglobulinemia (WM) cells to thrive. Additionally, the researchers will attempt to identify genes that allow WM cells to resist established therapies. The key is to identify specific gene targets that cause death of WM cells, but do not alter normal body cells. Any genes identified will be further tested in laboratory cells and then evaluated in mouse models. This research will hopefully identify new, previously unsuspected molecular targets for WM therapy.

FACTORS REGULATING IMMUNOGLOBULIN-PRODUCING B-CELLS IN PATIENTS WITH WALDENSTROM’S MACROGLOBULINEMIA – PART VI

TARGETING MYD88 SIGNALING IN WALDENSTROM’S MACROGLOBULINEMIA

This is a continuation of previous projects proposed by Dr. Treon and funded by the IWMF. In previous research partially funded by the IWMF, Dr. Treon and his team discovered the highly recurring mutation in the MYD88 gene that occurs in more than 90% of WM patients and showed that mutated MYD88 promoted growth and proliferation of WM cells through the downstream signaling pathways BTK and IRAK1/IRAK4. These findings enabled the pivotal clinical trial that led to approval of the BTK inhibitor ibrutinib (Imbruvica) for the treatment of WM in the US, Europe, and Canada. Resistance to ibrutinib is an emerging problem in WM patients, and Dr. Treon’s team has identified mutations in BTK that disrupt ibrutinib-BTK binding in samples from half of WM patients whose disease progressed on ibrutinib. His group has sought novel strategies to overcome the most common type of BTK mutation-related ibrutinib resistance in WM. His group is also working on uncovering the importance of other MYD88 downstream signaling pathways, including HCK, which triggers AKT, ERK1/2, and BTK itself. For this project, Dr. Treon has three principal Aims: 1) to delineate the importance of IRAK signaling to ibrutinib resistance and develop selective IRAK inhibitors based on this work, 2) to clarify whether HCK inhibition can suppress mutated BTK-acquired ibrutinib resistance in WM and develop selective HCK inhibitors, and 3) and to validate these inhibitors alone and in combination using animal models for future translation to clinical trials.

MYD88L265P SIGNALING-ASSOCIATED MULTIPLEX CHARACTERIZATION OF THE BONE MARROW MICROENVIRONMENT IN WM PATIENTS FOR CLINICAL APPLICATION

This project falls under the IWMF-LLS Strategic Research Roadmap Initiative. Dr. Carrasco will harness new and powerful digital pathology and artificial intelligence technology for better diagnostics and understanding of WM using two approaches. (1) More than 90% of WM patients have a mutation of the MYD88 gene, resulting in an altered protein, called MYD88 L265P. The altered MYD88 drives abnormal signaling which is key to survival and growth of WM cells. Detection of the MYD88 mutation by PCR is one of the cornerstones for making the diagnosis of WM. Dr. Carrasco has detected a feature of the mutant MYD88 protein, in which it forms microscopic aggregates inside cells. He proposes to detect these aggregates of mutant MYD88 protein with a technique called immunohistochemistry to speed up and simplify the diagnosis of WM. (2) WM cells live primarily in bone marrow, where they interact with other bone marrow cells. Some of the bone marrow cells help maintain a favorable environment for the WM cells to grow, while other bone marrow cells attack the WM cells and prevent excessive growth. Dr. Carrasco’s group, in collaboration with Drs. Treon, Hunter, and other state-of-the-art labs at Harvard, will examine bone marrow samples using automated digital pathology to identify WM cells and the specific cell types in their immediate proximity which may be interacting with the WM cells. He hypothesizes that the local interactions of WM cells with other bone marrow cells changes during disease progression and if disease resistance develops. If scientists could understand these local interactions between cells better, it may be possible to treat patients with drugs to make the bone marrow less supportive of WM cell growth or to enhance immune attack on the WM cells.

GENOMIC AND IMMUNE BIOMARKERS OF PROGRESSION FROM IGM MGUS TO WALDENSTROM’S MACROGLOBULINEMIA

A common benign precursor to WM is called IgM-MGUS (monoclonal gammopathy of undetermined significance). Of all the people with IgM-MGUS, a small proportion will progress to WM. Many patients with WM are first detected before they show significant disease symptoms. The disease at this stage is called “asymptomatic WM” or “smoldering WM.” The patients are monitored without being treated, a strategy called “watch-and-wait.” Some IgM-MGUS or watch-and-wait WM patients never progress to symptomatic WM or only progress after many years. Others progress more rapidly to active, symptomatic disease that requires treatment. Dr. Sklavenitis-Pistofidis’s project is designed to more accurately predict which patients will progress to active disease. He will build upon a predictive model that his group published earlier, which used clinical biomarkers such as the degree of bone marrow infiltration and the level of abnormal serum IgM. In the present proposal, Dr. Sklavenitis-Pistofidis will use advanced technologies to characterize patients’ tumor cells and immune cells. He will then incorporate genomic and immune information into an improved, next-generation predictive model. The model could be used as the foundation for a clinical trial to test whether the patients predicted to progress rapidly would benefit from earlier drug treatment. This is a Kyle Young Investigator Award project.

ISOLATION AND SPATIAL CHARACTERIZATION OF 6Q DELETIONS AND CXCR4 MUTATIONS USING NOVEL BIOMARKERS IN WM

Cell division isn’t a perfect process. When an original cell duplicates its DNA in order to divide into two new cells, it sometimes makes mistakes. Some of these mistakes, called mutations, can be passed on to the next generation of cells. As cells with the mutation continue to divide and make new cells, eventually, there are a large number of cells that share the same mutation. These are called clones. But cells in a clone keep dividing. Eventually, one of the cells in a clone makes a mistake in a different area of DNA and develops a new mutation. Soon, some of the cells from the original clone—but not all of them—have the new mutation in addition to the original mutation. This is called a subclone. The existence of different subclones makes WM complicated. In most WM patients, the MYD88 gene is mutated. However, if a scientist analyzes every one of the WM cells in a patient’s body, they may find some WM cells that also have a second mutation, such as a mutation in a particular part of the CXCR4 gene. In some patients, there are subclones of different CXCR4 mutations. Other MYD88 subclones may have other DNA modifications, such as deletions of a certain area of DNA called “6q” which contains a large number of genes. Dr. Guerrera’s project is to study subclones in WM patients. If a patient has multiple subclones, each with different mutations or other DNA alterations, how does that affect the disease? Does one subclone of WM cells influence the growth of another separate subclone of WM cells? How can different subclones be separately identified in the best way, when they are all mixed together in the same patient’s body? One of Dr. Guerrera’s goals is to identify new biomarkers that laboratories can use to identify and isolate different subclones within a patient. She will also investigate treatment—if a drug kills WM cells from one subclone, what about the other subclones? This work may lead to combination treatments, tailored to individual patients and intended to kill multiple subclones at the same time. This is a Kyle Young Investigator Award project.

HARNESSING EPIGENETIC SIGNATURES FOR NEW INSIGHT INTO THE MECHANISMS AND CLASSIFICATION OF WALDENSTROM MACROGLOBULINEMIA

DNA contains a specific code. When the code is read, it enables cells to make proteins. However, all the DNA in a cell is not read at any one time. Modifications—called “epigenetics”—regulate which parts of the DNA are available to the cell and which parts are not. Epigenetic modifications can make regions of DNA “open,” meaning that the genes in those regions are available to encode proteins for the cell, while other regions of DNA are “closed,” meaning that the genes are unavailable to the cell. Cancer cells often have unique patterns of epigenetic changes. This can cause cancer cells to express many proteins in an abnormal way, which can drive the cells to divide and grow more than they should. Dr. Oakes previously discovered two types of epigenetic changes in WM cells. Some WM patients had WM cells with a epigenetic pattern resembling a normal cell type called a plasma cell. Other WM patients had WM cells with a pattern resembling another normal cell type, called the memory B cell. In the present grant, Dr. Oakes will dig more deeply into understanding the epigenetic changes of WM cells, including the two types he previously identified. He will also try to better understand exactly what leads to the characteristic epigenetic changes seen in WM cells. It is well known that most WM patients have a mutated (altered) protein called MYD88. It is possible that specific signals from the abnormal MYD88 cause the epigenetic changes of WM cells. If scientists could better understand the epigenetic changes that regulate WM cells, it may be possible to use epigenetics-modifying drugs to restore normal regulation and control WM. This project falls under the IWMF-LLS Strategic Research Roadmap Initiative.

CHARACTERIZATION OF GENOMIC ALTERATIONS IN TREATMENT NAIVE PATIENTS WITH WALDENSTROM’S MACROGLOBULINEMIA THROUGH A COURSE OF TARGETED TREATMENT AND DISEASE PROGRESSION”

A MULTI-OMICS APPROACH FOR DECIPHERING THE MECHANISMS OF PROGRESSION IN PREMALIGNANT IGM GAMMOPATHIES: NEW INSIGHTS FROM THE FIL “BIO-WM” TRIAL

Recently, the IWMF established the Robert A. Kyle Career Development Award to encourage and fund research from young investigators, with the goal of generating the next generation of WM researchers.  Simone Ferrero, MD, is an Assistant Professor of Hematology at the University of Torino in Italy.  In this project, he will investigate the progression to symptomatic WM.  To do this, he will leverage samples already collected in the joint Italian-Spanish FIL BIO-WM clinical trial, in which several hundred people with IgM-Monoclonal Gammopathy of Uncertain Significance (IgM-MGUS), a common, non-neoplastic disorder, or smoldering WM (watch-and-wait WM) were repeatedly sampled over time to see what changes occurred in their cells and how those changes correlated with progression to overt WM.  While some gene mutations such as MYD88 and CXCR4 have already been characterized, Dr Ferrero will investigate other possible gene mutations. He will also evaluate RNA markers that might be diagnosed from blood samples, which would reduce the need for bone marrow biopsies.  The third part of the project is to study clonal hematopoiesis of indeterminate potential (CHIP), a common occurrence in older people, in which non-cancerous cells from the bone marrow harmlessly accumulate in the body.  Dr Ferrero will investigate genetic changes in these cell populations that might tip them over the edge to become harmful.  By making good use of the large number of patient samples that have already been collected from the FIL BIO-WM study, Dr Ferrero’s project may provide new insights into the underlying molecular changes that initially lead to development of WM. This could open new routes of therapy.

SINGLE-CELL MULTIOMICS FOR MINIMALLY INVASIVE ASSESSMENT OF TREATMENT EFFICACY IN WALDENSTROM’S MACROGLOBULINEMIA

ANALYSIS OF MOLECULAR AND MICROENVIRONMENTAL LANDSCAPE AND ITS ROLE IN DRUG RESISTANCE IN WALDENSTRÖM MACROGLOBULINEMIA

In an effort to encourage creative, innovative thinking, the IWMF initiated a new grant called the “Research Seed Money Initiative Grant.”  This is a one-year grant that allows a scientist to test a new idea.  If the project is successful, the scientist is then able to use the results to support a larger, multi-year grant to do a more in-depth investigation.  Damien Roos-Weil, MD, PhD is a clinical professor of hematology at the Pitié-Salpêtrière Hospital in Paris, France who has published some novel and highly impactful WM research recently.  WM cells mainly live in the bone marrow.  An emerging theme in WM research is that the bone marrow is not just a location where WM cells happen to grow.  Instead, different kinds of normal bone marrow cells associate and communicate with WM cells, forming a complex interaction that is necessary to allow WM cells to live and grow in the bone marrow.  Scientists are just beginning to unravel how different types of normal bone marrow cells encourage the growth of WM cells and what types of signals they use to do so.  Collectively, this complicated relationship is called the tumor microenvironment.  In this project, Dr Roos-Weil proposes to use bone marrow samples from several hundred WM patients from an on-going multinational European clinical trial and to apply new technologies to better understand the tumor microenvironment that allows WM cells to grow.  Drugs targeted against specific signaling pathways or cell types in the bone marrow present an opportunity for better combination WM drug therapy in the future.

TARGETING AN IRF4/CXCR4 AXIS TO REVERSE DRUG RESISTANCE IN WALDENSTROM’S MACROGLOBULINEMIA.

In an effort to encourage creative, innovative thinking, the IWMF initiated a new grant called the “Research Seed Money Initiative Grant.”  This is a one-year grant that allows a scientist to test a new idea.  If the project is successful, the scientist is then able to use the results to support a larger, multi-year grant to do a more in-depth investigation.  Leslie Crews, PhD, is an Assistant Professor of medicine at the University of California, San Diego with an interest in stem cell biology and blood cancers.  Dr Crews hypothesizes that abnormal expression inside cells of a regulatory protein called IRF4 (interferon regulatory factor-4) is involved in WM.  IRF4 is a key protein that drives early B cells to develop into more mature B cells and plasma cells.  When expressed in excessive amounts, IRF4 is important in cell survival in multiple myeloma, a related type of blood cancer.  If multiple myeloma cells express large quantities of IRF4, they can become drug-resistant.  Moreover, high levels of IRF4 can increase expression of CXCR4, a key protein in many people with WM.  Dr Crews will try to bridge these findings in multiple myeloma to WM, by asking if IRF4 is involved in WM cell proliferation, drug resistance, and CXCR4 over-expression.  Importantly, if she shows that IRF4 is involved in WM, her findings could quickly translate from the laboratory to human clinical trials.  A drug targeting IRF4 has been developed and is currently in clinical trials for multiple myeloma.  If Dr Crews Seed Money project successfully shows that IRF4 is involved with WM, she will test the effects of the new IRF4 drug in a mouse WM model.  Positive findings could lead to clinical trials of this novel drug in WM patients.

ANALYSIS OF THE CHROMATIN ACCESSIBILITY LANDSCAPE AND REGULATORY NETWORKS OF IgM MONOCLONAL GAMMOPATHIES: TOWARDS A BETTER UNDERSTANDING OF PROGRESSION MECHANISMS

In an effort to encourage creative, innovative thinking, the IWMF initiated a new grant called the “Research Seed Money Initiative Grant.”  This is a one-year grant that allows a scientist to test a new idea.  If the project is successful, the scientist is then able to use the results to support a larger, multi-year grant to do a more in-depth investigation.  While many impactful studies have been performed to identify mutations in key genes that lead to WM, there has been less attention to underlying gene regulation.  David Moreno, MD, is a hematologist at the Clinical and Provincial Hospital of Barcelona in Spain.  In this project, Dr Moreno will use new, state-of-the-art technologies to investigate what determines whether the expression level of genes is increased or decreased.  Cells use regulatory proteins, which attach to DNA, to determine when genes should be active or expressed.  However, DNA is arranged in a tightly packed conformation, and only certain areas of the DNA are exposed to the regulatory proteins.  This 3-dimensional packing varies from one cell type to another, resulting in different types of cell regulation, depending on the cell.  Specifically, using B cells and plasma cells, Dr Moreno will examine long stretches of DNA and determine where the DNA is exposed and accessible to regulatory proteins.  These techniques will be applied to cells from people with IgM MGUS (a precursor to WM), smoldering WM (also called asymptomatic or watch-and-wait WM), and symptomatic WM.  Results will be analyzed by computer to better understand the network of abnormal gene expression associated with progression to WM.  Better understanding of why cells progress from a benign precursor condition, IgM MGUS, to symptomatic WM may reveal new therapeutic targets for treatment.

USING MUTOGRAPHS TO DEFINE THE MOLECULAR LANDSCAPE AND CELL OF ORIGIN OF WALDENSTROM’S MACROGLOBULINEMIA

While substantial attention has been given to mutations of DNA that encode genes such as the well-known MYD88 or CXCR4, less work has been done on non-coding genome sequences, the sections of DNA located in-between genes.  These non-coding sequences are often involved in gene regulation.  Dr Gareth Morgan is a well-established Professor at the New York University Grossman School of Medicine with considerable experience in understanding the genetic basis of multiple myeloma.  He has accumulated a large set of DNA sequence data from WM patients, through multiple collaborations with other WM researchers.  He will use this data to look for variations in the non-coding regions.  He will combine these results with another test that can determine which areas of DNA are physically open and available to the cell machinery and which areas of DNA are covered up and inaccessible.  He will also use the DNA information to trace the multiple routes by which WM cells developed in the body.  Most gene mutations are harmless and do not cause cancer or other illnesses.  As a person gets older, these harmless “passenger mutations” accumulate as cells divide and form new cells, leaving an indelible history in the cells.  These mutational patterns are called “mutographs”.  Dr. Morgan will use computer systems that he and his group originally developed for the study of related cancers such as multiple myeloma.  With these computer tools, he can assemble and trace cells’ mutographs to reveal alternative pathways by which normal cells developed into WM cells.  It is increasingly obvious that there is no one treatment that will work on all people with WM.  Better understanding of the different types of WM, together with understanding the routes by which normal cells develop into WM, will hopefully lead to more precise, individualized WM treatments.  This project falls under the IWMF-LLS Strategic Research Roadmap Initiative.