Michael Churchill

Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox–5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.

ZFX controls propagation and prevents differentiation of acute T-lymphoblastic and myeloid leukemia.

Tumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

IL-17 producing mast cells promote the expansion of myeloid-derived suppressor cells in a mouse allergy model of colorectal cancer

Food allergy can influence the development of colorectal cancer, although the underlying mechanisms are unclear. While mast cells (MC) store and secrete histamine, immature myeloid cells (IMC) are the major site of histidine decarboxylase (HDC) expression, the enzyme responsible for histamine production. From our earlier work, we hypothesized that histamine is central to the association between allergy and colorectal carcinogenesis through its influence on the MC-MDSC axis. Here, we show that in wild type (WT) mice, ovalbumin (OVA) immunization elicits a typical TH2 response. In contrast, in HDC−/− mice, the response to OVA allergy is skewed towards infiltration by IL-17 expressing MCs. This response is inhibited by histamine treatment. The HDC−/− allergic IL-17-expressing MCs promote MDSC proliferation and upregulation of Cox-2 and Arg-1. OVA allergy in HDC−/− mice increases the growth of colon tumor cells in both the MC38 tumor cell implantation model and the AOM/DSS carcinogenesis model. Taken together, our results show that histamine represses IL-17-expressing MCs and their subsequent activation of MDSCs, attenuating the risk of colorectal cancer in the setting of food allergy. Targeting the MC-MDSC axis may be useful for cancer prevention and treatment in patients, particularly in those with food allergy.

Hematopoietic stem cell dysfunction underlies the progressive lymphocytopenia in XLF/Cernunnos deficiency

XRCC4-like factor (XLF/Cernunnos) is a component of the nonhomologous end-joining (NHEJ) pathway of double-strand DNA break repair. XLF-deficient patients develop a severe progressive lymphocytopenia. Although NHEJ is required for V(D)J recombination and lymphocyte development, XLF-deficient mice have normal V(D)J recombination, highlighting the need for an alternative mechanism for the lymphocytopenia. Here, we report that XLF-deficient mice recapitulate the age-dependent lymphocytopenia of patients. We show that XLF deficiency leads to premature aging of hematopoietic stem cells (HSCs), measured by decreased functional capacity in transplantation assays, preferential myeloid reconstitution, and reduced self-renewal at a young age. We propose that premature aging of HSCs, together with previously reported defects in class-switch recombination and memory immune response, underlies the progressive and severe lymphocytopenia in XLF-deficient patients in the absence of measurable V(D)J recombination defects.

Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

ABSTRACT The colorectal tumor microenvironment contains a diverse population of myeloid cells that are recruited and converted to immunosuppressive cells, thus facilitating tumor escape from immunoediting. We have identified a genetically and functionally distinct subset of dynamic bone marrow myeloid cells that are characterized by histidine decarboxylase (HDC) expression. Lineage tracing in Hdc-CreERT2;R26-LSL-tdTomato mice revealed that in homeostasis, there is a strong bias by HDC+ myeloid cells toward the CD11b+Ly6Ghi granulocytic lineage, which was accelerated during azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colonic carcinogenesis. More importantly, HDC+ myeloid cells strongly promoted colonic tumorigenesis, and colon tumor progression was profoundly suppressed by diphtheria toxin A (DTA)-mediated depletion of HDC+ granulocytic myeloid cells. In addition, tumor infiltration by Foxp3+ regulatory T cells (Tregs) was markedly impaired following HDC+ myeloid cell depletion. We identified an HDC+ myeloid-derived Cxcl13/Cxcr5 axis that mediated Foxp3 expression and Treg proliferation. Ablation of HDC+ myeloid cells or disruption of the Cxcl13/Cxcr5 axis by gene knockdown impaired the production and recruitment of Tregs. Cxcl13 induction of Foxp3 expression in Tregs during tumorigenesis was associated with Stat3 phosphorylation. Overall, HDC+ granulocytic myeloid cells affect CD8+ T cells directly and indirectly through the modulation of Tregs and thus appear to play key roles in suppressing tumoricidal immunity.

The ZFX Target Gene, FAM92A1, Is a Marker of AML Aggressiveness

The propagation of leukemia requires activation of stem cell transcriptional programs that support long term self-renewal. Previously we showed that the stem cell transcription factor ZFX is critically important for propagation of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). We have identified ZFX target genes in mouse and human leukemia cells using gene expression profiling in conjunction with chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq). Some ZFX target genes have a well established role in controlling mitochondrial function and cell metabolism, suggesting that ZFX is required, in part, to maintain metabolic homeostasis in leukemia propagating cells. The biological functions of other ZFX target genes have not been well characterized. One such gene, FAM92A1, is heavily dependent on ZFX for its expression in mouse and human AML cells. Genetic deletion of ZFX in mouse AML and lentiviral RNAi mediated knockdown of ZFX in human AML cell lines markedly decreased FAM92A1 expression. ChIP-seq showed a ZFX binding site in a dense CpG island between the first and second exons, which was confirmed by ChIP-PCR and suggests that ZFX directly controls FAM92A1 transcription at the proximal promoter. Our studies in sorted murine AML cells and human AML patient samples demonstrate that FAM92A1 is preferentially expressed in the leukemia initiating cell-enriched fractions. Analysis of large-scale gene expression studies of human AML patients revealed that high FAM92A1 expression is associated with the Flt3-ITD mutation, high-risk cytogenetics, early AML recurrence, and patient death. Multivariable analysis showed that it was an independent risk factor for shortened progression-free survival. In addition, FAM92A1 expression is increased in patients with myodysplastic syndrome-refractory anemia with excess blasts. These findings identify FAM92A1 as a marker for ZFX transcriptional activity and AML aggressiveness. The increased expression of FAM92A1 in AML with Flt3-ITD and high-risk cytogenetics suggests that it may be a functionally important component of the ZFX-driven self-renewal transcriptional program in high-risk AML. Definitive functional studies are warranted to establish the role of FAM92A1 in AML propagation.

Abstract 2425: An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS)

Background: The pathophysiology of low-risk myelodysplastic syndrome (LR-MDS) remains unknown and therapeutic options are limited. In a phase-II trial, with oral rigosertib, a small molecule inhibitor of PI3K and PLK pathways, was shown to have a 39% response rate, in patients with LR-MDS. Rigosertib caused an increase in hemoglobin, a decrease in transfusion requirements, and, in some patients who were previously refractory, “re-sensitization” to erythropoietin (EPO) therapy. Thus far, the mechanism of this responsiveness remains unknown. Whole exome sequencing revealed broad spectrum mutations in these patients, including mutations in SF3B1, SRSF2 and TET2. However, there was no correlation between mutational spectrum and responsiveness. Methods: CD34+ cells were isolated from bone marrow (BM) mono nuclear cells derived from BM aspirates. Bone marrow stromal cells (BMSC) were isolated from bone biopsies of patients with MDS. Normal CD34+ cells and BMSC were obtained commercially. CD34+ cells were co-cultured with stromal cells plated a day before and stimulated with concentrations of EPO and rigosertib. Differentiation was assessed by staining cells with various markers of erythroid differentiation and analyzed by FACS. We show that co-culture of CD34+ stem cells from the bone marrow of patients with their own BMSC recapitulates key features of the MDS phenotype and rigosertib responsiveness. Results: Normal (non-MDS) CD34+ cells co-cultured with normal BMSC showed striking responsiveness to EPO stimulation, as evidenced by the increased production of CD45- low/GPA+/ band 3+/Integrin-Alpha4+ erythroid cells (2.47 fold increase compared to no EPO). In contrast, RARS CD34+ cells co-cultured with MDS stromal cells showed no erythroid differentiation with EPO stimulation (1.09 fold change compared to EPO). Notably, LR-MDS co-cultures showed increased erythroid differentiation with the addition of 20 nm and 100 nm rigosertib in the presence of EPO (1.28 and 1.4 fold increase, respectively, compared to EPO alone). Moreover, co-cultures obtained from 3/3 patients that showed responsiveness in vivo were responsive to combined EPO/rigosertib stimulation in vitro, while 2/2 non-responsive patients were not responsive in vitro. Conclusions: We have created an in vitro platform to dissect the mechanism of rigosertib responsiveness in RARS patients. This is a novel co-culture-based system that recapitulates key features of the RARS phenotype. Without reliable animal models for this disease, this platform may offer a viable method to characterize drug responsiveness, dissect mechanisms, and offer patient-specific drug responsiveness information, since individual CD34+ cells are co-cultured with a patient9s own BMSC. We wish to use this in vitro co- culture test to prospectively predict responsiveness to experimental drugs in patients, thereby focusing the drug on only selected patients. Citation Format: Daniela Georgieva, Sheherzad Preisler, Michael Churchill, Abdullah Mahmood Ali, Azra Raza, Siddhartha Mukherjee. An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2015-2425