RauhLab

Department of Pathology and Molecular Medicine

RauhLab

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Goals

Our goals are to improve the diagnosis, prognosis and treatment of patients with myeloid cancers and to manipulate myeloid innate immune cells for cancer and transplant immunotherapy.

Research Directions

I) Translational & Transformative Myeloid Cancer Pathology - MDS

I) Translational & Transformative Myeloid Cancer Pathology - MDS

photo from overhead of hands adding vials to a centrifuge in pathology labMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic neoplasms, manifesting with dysplastic bone marrow failure, peripheral blood cytopenias and variable predisposition for transformation to acute myeloid leukemia (AML).  Diagnosis is heavily reliant on non-specific, subjective morphology, particularly in early stages – karyotype results are most often normal, and no MDS-specific molecular assays currently existDiagnosis is often delayed, requiring serial invasive bone marrow investigations.  Moreover, MDS primarily afflicts older adults and, without molecular targets and assays, treatments are mainly supportive (i.e. blood transfusions and growth factors).  While some MDS patients respond to emerging agents, such as hypomethylating agents, it is not entirely clear how to predict treatment responses.  With our aging demographics, MDS prevalence is expected to increase, along with demands for novel and more personalized treatment.  

Recurring MDS-associated somatic mutations and gene expression signatures have recently been discovered that convey diagnostic and prognostic information not captured with current pathology platforms.  In order to address these shortcomings, we have assembled clinically-rich MDS databases, bone marrow banks and clinical collaborators at Sunnybrook Health Sciences Centre (Drs. Richard Wells and Rena Buckstein) and Kingston General Hospital (Drs. John Matthews, David Lee and colleagues), and are poised to access bone marrow specimens from an NCIC/SWOG MDS clinical trial (Dr. Lois Shepherd).  Moreover, we have established at Queen’s University a core Ion Torrent genomic sequencing facility (in conjunction with Drs. Harriet Feilotter and Paul Park), a CFI-funded NanoString nCounter gene expression analysis platform, and the histopathology resources of the Queen’s Laboratory for Molecular Pathology (in conjunction with Dr. David LeBrun).

Recently, this research arm has received generous funding support from the Transformative Pathology Division of the Ontario Institute of Cancer Research (OICR), and a Canadian Cancer Society Research Institute Innovation (CCSRI) Grant.

Hypotheses:

  1. Patients aged > 65 will have detectable MDS-associated mutations in their peripheral blood.
  2. Patients aged > 65 with any or all of unexplainable cytopenia(s), macrocytosis or anisocytosis will have a higher frequency of these MDS mutations and the Sunnybrook MDS risk score will identify those with an increased risk (odds ratio) of detectable MDS mutations.
  3. MDS somatic mutation profiling will also:
    1. Reveal cryptic MDS-associated mutations in patients with equivocal bone marrow investigations preceding WHO-standard, MDS-diagnostic marrows
    2. Predict treatment responses to the hypomethylating agent, azacytidine
    3. Provide prognostic information not captured by current MDS clinical risk score
  4. NanoString nCounter-based gene expression profiling will provide prognostic information not captured by current MDS clinical risk scores.​

Specific Aims:

  1. Diagnostic: The gene mutation panel may provide a more objective molecular test that can identify clonal versus reversible, non-clonal causes of dysplasia, leading to closer clinical monitoring and earlier identification of potentially targetable driver mutations. Together, these may lead to earlier and less invasive detection of MDS.
  2. Prognostic: The gene mutation panel and NanoString nCounter-based digital mRNA expression assay may contribute to refined clinical risk classification of MDS patients, and may eventual help to tailor patient/risk-specific treatment.
  3. Predictive: The mutation panel includes genes involved in epigenetic regulation and preliminary evidence suggests this may help predict which patients will benefit from hypomethylating agents and which patients should be spared unnecessary side effects.

Photo of two NanoString machines on a lab bench at Rauh Lab

II) The Immune Environment of MDS – M2 Macrophages and MDSCs

II) The Immune Environment of MDS – M2 Macrophages and MDSCs

M2-macrophages (M2-MΦ) and more immature, M2-like, myeloid-derived suppressor cells (MDSC) are expanded by solid cancers and exploited for their immunosuppressive properties, including arginase 1 (Arg1)-mediated T-cell inactivation.  Our group and others have discovered a regulatory circuit downstream of GM-CSF and IL-3, involving Lyn, Hck, and SHIP1, acting at the level of the “SPS complex” (Shp-1/PLC-b3/Stat5) to dephosphorylate Stat5 and inhibit M2 transcriptional programs.  In genetic systems, SPS complex inactivation (i.e. SHIP1-/-, Shp-1MeV/MeV, Lyn-/-;Hck-/-, or Lyn-/-;PLC-b3-/-) leads to a disorder resembling myelodsyplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).  In keeping, we and others have described Arg1 over-expression and MDSC expansion in human CMML and low-risk MDS patients.  However, neither mutations in SHIP1 nor other SPS components are common in human patients.  Instead, the most recurrently mutated gene in these myeloid cancers is TET2, which encodes an epigenetic regulator of the methycytosine dioxygenase family.  Genetic inactivation of TET2 also closely resembles the MDS/CMML-like disorder seen with SPS complex-inactivation. 

Hypotheses: 

TET2 (and/or other) mutations impact common GM-CSF and IL-3 signaling pathways in CMML and MDS, defining a novel subtype of M2-skewed myeloid cancer.  TET2 (and/or other) mutation profiling will lead to earlier detection and refined classification of CMML and MDS patients.

Specific Aims:

Arginase 1 immunohistochemical staining of representative control, CMML and MDS patient bone marrow biopsies.

Figure – Arginase 1 immunohistochemical staining of representative control, CMML and MDS patient bone marrow biopsies.
  1. Address the lack of basic knowledge regarding the expression pattern and role of TET2 protein in myelomonocytic differentiation and activation.
  2. Characterize the effects of TET2 inactivation on MDSC and M2-MΦ compartments in and potential genetic complementarity of compound TET2 and SPS complex inactivation.
  3. Determine if TET2-mutations are associated with the M2-immune signature in human CMML and MDS and their translation potential to improve myeloid cancer detection and classification.

 

III) Harnessing M2-Macrophages/MDSCs for Hemophilia Factor VIII Tolerance

III) Harnessing M2-Macrophages/MDSCs for Hemophilia Factor VIII Tolerance

Among FVIII-treated hemophilia A (HA) patients, 25-30% develop FVIII antibodies that inhibit its pro-coagulant function.  However, the etiology of FVIII inhibitor formation remains poorly understood, particularly the role played by myeloid innate immune cells.  Myeloid-derived suppressor cells (MDSC – CD11b/Gr1 co-expressing, immunosuppressive myeloid cells found in peripheral blood, lymphoid tissue and bone marrow) and M2-macrophages (M2-MΦ) are expanded in cancer and inhibit adaptive immune responses against tumors.  MDSC and M2-MΦ also mediate organ transplant tolerance.  Therefore, we are investigating MDSC and M2-MΦ dynamics during the course of FVIII exposure in HA systems and the potential to harness MDSC/M2-MΦ as a novel means of mediating FVIII tolerance.  These studies are being conducted with the collaboration of Dr. David Lillicrap and his research group.

Hypothesis: 

The immunosuppressive potential of MDSC/M2-MΦ may be harnessed for the prevention/treatment of inhibitory FVIII antibody formation.Photo of flow cytometer on lab bench in Rauh Lab

Specific Aims:

  1. Based on the emerging role of MDSC/M2-MΦ in transplant tolerance, and our preliminary evidence, investigate the role of endogenous MDSC in a Hemophilia A model of differential FVIII tolerance.
  2. Determine the in vivo therapeutic potential of ex vivo-generated murine MDSC from a clinically-applicable source (autologous bone marrow) to prevent inhibitory FVIII antibody production.
  3. Obtain further functional data for external grant support and build future capacity for translational human investigations of the role and therapeutic potential of MDSC in human HA FVIII tolerance.

Data from the flow cytometer

MDSC flow data