ALBERT

Description: Several studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2–related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2−/−) BM were not elevated compared with wild-type. Nrf2−/− HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2−/− BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2−/− HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2−/− cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

Description: Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and 〉60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Hedgehog (Hh) signaling is essential for normal development and is dysregulated in many cancers. Hh signaling is active in normal bone marrow and the majority of acute myeloid leukemias, however, the precise role of Hh signaling and its positive effector Gli1 in normal or malignant hematopoiesis is not known. We have analyzed the bone marrow of Gli1 null mice to understand the role of this transcription factor in normal hematopoiesis in order to gain insight into its potential role in leukemia. Gli1 null mice develop normally and have normal peripheral blood counts but the bone marrow shows skewing of the c-Kit+Sca1+Lin-neg (KSL) progenitor compartment with increased CD34negKSL long-term HSC (LT-HSC) and decreased 34+KSL short-term HSC (ST-HSC). An analogous difference was observed in the c-Kit+Sca1negLinneg (KL) myeloid progenitor compartment with an increase in FcRγlowCD34+KL common myeloid progenitors (CMP) and decrease in the FcRγhighCD34+KL granulocyte monocyte progenitors (GMP). We speculated that these differences could be due to impaired cell cycle since both the ST-HSC and GMP are more proliferative than LT-HSC and CMP, respectively. Cell cycle analysis by DNA content and BrdU pulse labeling (100mg/kg IP 14 hours prior to analysis) revealed a marked decrease of proliferation in the LT-HSC, ST-HSC, CMP, and GMP compartments of Gli1 null mice. We supported this conclusion by demonstrating that the bone marrow of Gli1 null mice are relatively radio-resistant. Mice exposed to 400 cGy of total body irradiation followed with serial blood counts revealed less severe nadir, but delayed rebound of white blood cells in Gli1 null mice. We further hypothesized that although Gli1 appears to be dispensable for steady-state peripheral hematopoiesis, it might be necessary for rapid proliferation of progenitors needed during stressed hematopoiesis. In brain development, where Hh signaling is much better understood, active Hh signaling is critical for regulating proliferation of neural stem cells and Gli1 activity significantly increases after depletion of neural progenitors with chemotherapy (Bai et al., Development, 2002). To extend this observation to hematopoiesis, we treated Gli1 null mice and wild-type litter-mates with 5-fluorouracil (5-FU) at 100mg/kg and measured serial blood counts. Gli1 null mice had a delayed recovery of total white blood cells and neutrophil counts at 6 days after 5-FU, but this difference normalized by 20 days after treatment. To confirm that this difference was due to impaired proliferation and not increased sensitivity to 5-FU, we treated Gli1 null and wild-type mice with G-CSF (10mcg/kg/day) for three days to stimulate neutrophil proliferation. Confirming our hypothesis, we observed an attenuated neutrophil response in G-CSF stimulated Gli1 null mice. In summary, we have demonstrated that Gli1 loss leads to decreased HSC and myeloid progenitor proliferation, which has important functional consequences for stress hematopoiesis. These data suggest that abnormal Hh activity in leukemia may be important for driving the uncontrolled proliferation of cancer cells. Gli1 null mice were a kind gift from Alexandra Joyner, Memorial Sloan-Kettering Cancer Center

Description: Aberrant self-renewal is a hallmark of cancer and is central to the initiation, maintenance and relapse of clinical disease. The cellular processes responsible for self-renewal have not been delineated in most human cancers, but it is likely that conserved pathways required for the regulation of normal stem cells are involved. Notably several highly conserved signaling pathways that regulate stem cell fate decisions during embryonic development, such as Notch, Hedgehog (Hh) and Wingless (Wnt), are inappropriately activated in a wide variety of human cancers. We recently demonstrated that the Hh signaling pathway is required for the maintenance of cancer stem cells in the plasma cell malignancy, multiple myeloma. Since myeloma stem cells phenotypically resemble normal B cells, we hypothesized that aberrant Hh signaling is a feature of other B cell malignancies. We studied established human cell lines derived from patients with classical Hodgkin lymphoma (L428, KM-H2), diffuse large B cell NHL (HT, Pfeiffer, RL, and Hs 602), and mantle cell NHL (Granta 519, Jeko-1, Rec-1) and found that expression of the Hh signaling pathway components PATCHED (PTCH), SMOOTHENED (SMO), and GLI1, 2 or 3 by RT-PCR was markedly elevated compared to normal B cells in the majority of cell lines from each subtype of lymphoma. In order to examine the functional role of Hh signaling on human lymphomas, cells were treated with recombinant human sonic Hh ligand (SHh) or the naturally occurring inhibitor of SMO, cyclopamine, followed by evaluation of clonogenic growth in methylcellulose. Resulting colony formation was significantly increased in response to activating SHh ligand, whereas treatment with cyclopamine significantly inhibited clonogenic recovery. Similarly, the inhibition of pathway signaling by neutralizing anti-Hh antibodies limited colony formation suggesting that ligand binding by PTCH was required for pathway activation similar to other non-Gorlins tumors such as small cell lung cancer, pancreatic carcinoma, and metastatic prostate cancer that lack mutation of pathway components. Furthermore, we examined the activity of a novel semi-synthetic cyclopamine analogue, IPI-609, and found that it also limited clonogenic lymphoma growth. The effects of IPI-609 were highly specific as the clonogenic recovery of cell lines lacking expression of Hh pathway components was not affected by treatment. Our previous studies in multiple myeloma have suggested that cancer stem cells can be identified by their relatively higher activity of the intracellular enzyme retinaldehyde dehydrogenase (ALDH) similar to normal hematopoietic and neural stem cells. We found that high ALDH activity could also identify rare cell populations with greater clonogenic growth potential compared to ALDHlow/neg cells in the majority of lines and treatment with cyclopamine or IPI-609 significantly reduced the relative proportion of ALDHhigh cells. Therefore, the Hh signaling pathway may represent a novel therapeutic target in human lymphomas. Moreover, novel Hh inhibitors, such as IPI-609, may inhibit highly clonogenic lymphoma cancer stem cells responsible for disease relapse.

Description: The Hedgehog (Hh) pathway is essential for normal embryonic development and tissue repair. The role of Hh signaling in hematopoiesis has been studied primarily by modulating the activity of Patched and Smoothened, but results have been conflicting. Some studies demonstrate a requirement for pathway activity in hematopoiesis, whereas others report that it is dispensable. Hh activity converges on the Gli transcription factors, but the specific role of these downstream effectors in hematopoiesis has not been reported. We have analyzed hematopoietic stem cell (HSC) and progenitor function in mice with a homozygous deletion of Gli1 (Gli1null). Gli1null mice have more long-term HSCs that are more quiescent and show increased engraftment after transplantation. In contrast, myeloid development is adversely affected with decreased in vitro colony formation, decreased in vivo response to granulocyte colony-stimulating factor (G-CSF), and impaired leukocyte recovery after chemotherapy. Levels of the proto-oncogene Cyclin D1 are reduced in Gli1null mice and may explain the loss of proliferation seen in HSCs and progenitor cells. These data demonstrate that Gli1 regulates normal and stress hematopoiesis. Moreover, they suggest that Gli1 and Smoothened may not be functionally redundant, and direct GLI1 inhibitors may be needed to effectively block HH/GLI1 activity in human disease.

Description: Key Points GLI3R inhibits Hh signaling and is required for response to SMO antagonist in AML. GLI3 is silenced in AML, and decitabine restores GLI3 expression and leads to modulation of Hh signaling.

Description: Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p 〈 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p

Description: Background: In a previously reported Phase 2 randomized study of patients with acute myeloid leukemia (AML), addition of the investigational agent glasdegib (PF-04449913) to low-dose cytarabine (LDAC) improved overall survival (OS) when compared with LDAC alone. In a non-randomized study arm, glasdegib together with 7+3 chemotherapy was well tolerated and associated with clinical activity. We used a comprehensive biomarker analysis, evaluating gene expression, circulating cytokine levels, and gene mutations, to identify molecular drivers that predict overall response (OR) and OS. Methods: In this Phase 2 multicenter study (NCT01546038), patients with AML who were suitable for non-intensive therapy were randomized (2:1) to LDAC + glasdegib 100 mg QD or LDAC alone, and patients suitable for intensive therapy were assigned 7+3 plus glasdegib 100 mg QD. Whole blood, serum, and bone marrow aspirate samples were collected at baseline, and used to assess 19 genes for expression analysis, 38 analytes for circulating cytokine levels, and 109 genes for mutation analysis. Gene expression was analyzed using TaqMan Low Density Array Cards (TLDCs), cytokine levels were analyzed using quantitative, multiplexed immunoassays (Myriad RBM), and mutation analysis was performed using the Illumina® MiSeq instrument (San Diego, CA). All correlations were performed either for OS or for OR. For gene expression and cytokine analysis, a cut-off value above or below the median expression level for each treatment arm was used to separate samples into two subgroups (〈 or ≥ the median value) to explore the relationship of expression levels with OS data. Criteria for significance in the non-intensive cohort required one subgroup to have a p-value of

Description: Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Although a majority of patients are cured with standard chemo-immunotherapy, up to 40% of DLBCL patients have refractory disease or develop relapse following R-CHOP regimen, warranting development of novel, more effective therapeutic strategies for this cohort[1]. The composition of immune cells in the tumor microenvironment (TME) and tumor PD-L1 expression have been reported to predict DLBCL outcomes, however PD1 inhibitors demonstrate response rates of less than 10%.[2-5] We hypothesize that a better characterization of spatial architecture of the tumour microenvironment (TME) in lymphoma will help explain why DLBCL has poor responses to PD1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [6]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for relevant immunophenotypes, the spatial architecture of those phenotypes and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype. Immune cells represented 33% of the cells broken down to CD4 (36%), CD8 (30%), macrophages (26%) and TREG(8%) (Figure A).Immune cell infiltration in individual tumor samples ranged from 7% to 75% with marked heterogeneity between samples. Analysis of immune marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure B-C) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macrophages, and tumor cells (Figure D). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure E). Each CD8 spatial interaction pattern is distinctive with case to case heterogeneity (Figures F). Risk assessment analyses of spatial clusters 1, 2 and 4 ("hazardous") had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 ("protective") (Figure G). In the "protective" spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREGphenotypes, with opposite in "hazardous" areas (Figures H). TIM-3 expression was high both on T cells and tumor cells in the "hazardous" neighborhoods. Finally, we show that sub-setting our analysis of CD8 phenotypes based on their spatial location to other cells improved our ability to predict overall survival in the cohort. Conclusion These results are the first to demonstrate that spatial profiling of immune architecture in DLBCL is associated with clinical outcomes, and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies. References [1] Coiffier, B. et al.,Blood116, 2040-5 (2010). [2] Lenz, G. et al.,N. Engl. J. Med.359, 2313-2323 (2008). [3] Ansell, S. et al.,J. Clin. Oncol.19, 720-726 (2001). [4] Qiu, L., et al., BMC Cancer19, 273 (2019). [5] Kridel, R., et al., Haematologica100, 143-5 (2015). [6] Giesen, C. et al., Nat. methods |11, 417 (2014). Figure Disclosures Merchant: Agios: Speakers Bureau; Pfizer: Consultancy, Research Funding.