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The dysregulation of developmental and stem cell–associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1–Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.Significance:We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin–MLL inhibition in NPM1c leukemias.See related article by Wang et al., p. 724.This article is highlighted in the In This Issue feature, p. 517

Supplementary Figure 1. NPM1c degradation leads to differentiation of OCI-AML3 cells.Supplementary Figure 2. NPM1c chromatin targets identified in OCI-AML3-NPM1c-FKBP12 cells and validated by targeted degradation.Supplementary Figure 3. Motif analysis reveals developmental transcription factor binding sites at NPM1c bound regions.Supplementary Figure 4. NPM1c chromatin targets are highly expressed in patient samples of NPM1c AML.Supplementary Figure 5. NPM1c degradation has rapid effects on transcription at it target genes.Supplementary Figure 6. Transcriptional machinery is lost from NPM1c targets after 1 hour of NPM1c degradation.Supplementary Figure 7. CRIPSR domain scan reveals specific dependency of NPM1 mutant cells on the AS2 region.Supplementary Figure 8. The AS2 region affects NPM1c localization in 293T and OCI-AML3 cells.Supplementary Figure 9. Selinexor treatment disrupts NPM1c chromatin binding.Supplementary Figure 10. NPM1c cooperates with MLL1 to drive oncogenic gene expression.Supplementary Figure 11. NPM1c is selectively lost from chromatin in response to Menin inhibitor treatment.Supplementary Figure 12. Menin-MLL inhibition leads to selective loss of NPM1c target gene expression.

The dysregulation of developmental and stem cell–associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1–Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.Significance:We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin–MLL inhibition in NPM1c leukemias.See related article by Wang et al., p. 724.This article is highlighted in the In This Issue feature, p. 517

Table S1. Read normalized ratios of ChIPseq signal of untreated versus dTAG-13 treated OCI-AML3-NPM1c-FKBP12 cells.Table S2. Summary of NPM1c and NPM1 Chromatin Binding Targets.Table S3. Patient info for patient derived xenograft (PDX) models used.Table S4. ChIPseq reads per kilobase (rpk) data summary of PDX samples with NPM1c-Ab and NPM1-Ab.Table S5. RNAseq differential expression data of OCI-AML3-NPM1c-FKBP12 treated with dTag-13 for 3 or 24 h and OCI-AML3 cells treated with Selinexor for 6 h.Table S6. SLAMseq differential expression data of OCI-AML3-NPM1c-FKBP12 cells treated with dTag-13 for 15, 30, 60, 120 or 180 minutes.Table S7. PROseq differentially transcribed genes in OCI-AML3-NPM1c-FKBP12 cells treated with dTag-13 for 30 or 60 minutes and OCI-AML3 cells treated with VTP-50469 for 48 hours.Table S8. CRISPR tiling screen with NPM1 WT specific guides (blue) and NPM1c specific guides (red).Table S9. ChIPseq rpk values for CRM1 and HA ChIPs in 293T cells.Table S10. RNAseq differential gene expression analysis in 293T cells after overexpression of NPM1c-HA compared to all other samples (MIG, NPM1wt-HA and AS1-HA).Table S11. Fold change in ChIPseq differential peak interval signal in OCI-AML3 cells treated with Selinexor (100 nM, 6 hours) or VTP-50469 (330 nM, 96 hours).Table S12. Oligos used in this study.Table S13. Antibodies used in this study.Table S14. Summary of total and mapped reads of ChIPseq samples.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Supplementary Figure from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

Supplementary Figure S1 shows the cellular effects of AKIs and anti-leukemic agents across a panel of human AML cell lines. Supplementary Figure S2 shows expression of aurora-A and aurora-B protein levels in four AML cell lines. Supplementary Figure S3 shows AMG 900 induces a dose-dependent increase in polyploidy, apoptosis, and p53 protein levels in MOLM-13 cells. Supplementary Figure S4 shows AMG 900 plus Ara-C combination matrix and CI determination in MOLM-13 cells. Supplementary Figure S5 shows AMG 900 induced apoptosis is attenuated by peptide inhibitors of caspases in MOLM-13 cells. Supplementary Figure S6 shows FC gating strategy for annexin-V coupled JC-1 assay. Supplementary Figure S7 shows the anti-proliferative effects of AMG 900 and AZD1152-hQPA on primary human bone marrow mononuclear cells in culture. Supplementary Figure S8 shows a moderate reduction in mouse body weight after AMG 900 treatment.

Supplementary Table from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.Significance:Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307

Purpose:The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear.Experimental Design:We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage–committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN.Results:JAK2V617F-mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis.Conclusions:Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Supplementary figures and figure legends

Purpose:Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis.Patients and Methods:Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis.Results:In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients.Conclusions:Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868

Figure S1: Alisertib treatment did not result in a consistent cytokine response. Figure S2: Alisertib did not have a consistent effect on TGF-b levels. Figure S3: Alisertib induced GATA1 expression in the SET2 megakaryocytic cell line. Figure S4: AURKA inhibition induces megakaryocytic gene expression. Figure S5: Alisertib increases GATA1 expression in the bone marrow of patients on therapy. Table S1: Correlation of Genotype and prior JAK inhibitor therapy with response and adverse events Table S2: Summary of Treatment Related Adverse Events Table S3: Association between GATA1 staining, degree of fibrosis and clinical response

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

Purpose: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. Experimental Design: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage–committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. Results: JAK2V617F-mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Conclusions: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Purpose: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. Patients and Methods: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. Results: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. Conclusions: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis. See related commentary by Piszczatowski and Steidl, p. 4868

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. Abstract: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

Human cytomegalovirus (HCMV) establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to the establishment of latency in these cells. The early events in infection may have a critical role in shaping the establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times postinfection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of polymerase II (Pol II), histone 3 lysine 27 acetylation (H3K27Ac), and histone 3 lysine 27 trimethylation (H3K27me3) occupancy of the viral genome showed that (i) Pol II occupancy was highest at the major IE promoter (MIEP) at 4 h postinfection. However, it was observed throughout the genome. (ii) At 24 h, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication oriLyt; (iii) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac but not with changes in chromatin accessibility or a switch in modification of H3K27. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen in immunocompromised hosts and developing fetuses. Current antiviral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

The myeloproliferative neoplasms frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis and an accumulation immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial ROS and stabilization of HIF1a, and downregulation of LKB1 and increased levels of HIF1a were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated. We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs. Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G). In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF. 1. Tefferi A, et al. J Clin Oncol (2011) 2. Fisher DAC, et al. Leukemia (2019) 3. Chen P, et al. Front Med (2021) Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

The depletion of tumor-associated macrophages (TAMs), involved in different stages of cancer development and progression, is an appealing strategy in cancer therapy. We developed novel Clodronate-containing liposomes (Clo-Lipo-DOTAP) presenting physicochemical properties (size distribution, polydispersity index and Z-potential) suited for safe storage and injections. In vitro, Clo-Lipo-DOTAP inhibited proliferation, reduced viability and induced apoptosis of a macrophage-like cell line in a dose- and time-dependent manner. In proof of functionality experiments, Clo-Lipo-DOTAP depleted macrophages in a genetic mouse model of chronic hepatitis and hepatocellular carcinoma leading to a significant reduction of F4/80-positive cells in the liver and spleen of treated mice compared to PBS-treated controls. The number of granulocytes, B and T lymphocytes was not affected. In B16/F10 subcutaneous melanoma-bearing mice, Clo-Lipo-DOTAP significantly reduced the volume of primary tumors (P In conclusion, the depletion of TAMs in primary and metastatic melanoma presents anti-tumor efficacy via inhibition of angiogenesis and modulation of inflammation related cytokines.

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

The presence of interval sentinel lymph nodes in melanoma is documented in several studies, but controversies still exist about the management of these lymph nodes. In this study, an immunohistochemical evaluation of tumor cell proliferation and neo-angiogenesis has been performed with the aim of establishing a correlation between these two parameters between positive and negative interval sentinel lymph nodes. This retrospective study reviewed data of 23 patients diagnosed with melanoma. Bioptic specimens of interval sentinel lymph node were retrieved, and immunohistochemical reactions on tissue sections were performed using Ki67 as a marker of proliferation and CD31 as a blood vessel marker for the study of angiogenesis. The entire stained tissue sections for each case were digitized using Aperio Scanscope Cs whole-slide scanning platform and stored as high-resolution images. Image analysis was carried out on three selected fields of equal area using IHC Nuclear and Microvessel analysis algorithms to determine positive Ki67 nuclei and vessel number. Patients were divided into positive and negative interval sentinel lymph node groups, and the positive interval sentinel lymph node group was further divided into interval positive with micrometastasis and interval positive with macrometastasis subgroups. The analysis revealed a significant difference between positive and negative interval sentinel lymph nodes in the percentage of Ki67-positive nuclei and mean vessel number suggestive of an increased cellular proliferation and angiogenesis in positive interval sentinel lymph nodes. Further analysis in the interval positive lymph node group showed a significant difference between micro- and macrometastasis subgroups in the percentage of Ki67-positive nuclei and mean vessel number. Percentage of Ki67-positive nuclei was increased in the macrometastasis subgroup, while mean vessel number was increased in the micrometastasis subgroup. The results of this study suggest that the correlation between tumor cell proliferation and neo-angiogenesis in interval sentinel lymph nodes in melanoma could be used as a good predictive marker to distinguish interval positive sentinel lymph nodes with micrometastasis from interval positive lymph nodes with macrometastasis subgroups.

Background/Aims: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. Methods: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell’s apoptosis, and HG3 transplant mice survival with drug treatment. Results: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. Conclusion: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of Non-Hodgkin lymphoma characterized by clinical and biological heterogeneity attributable both to the tumor cells and the complex tumor-microenvironment surrounding them. Tumor-associated macrophages (TAMs) and mast cells are two major components of the tumor inflammatory infiltrate with a definite role in enhancing tumor angiogenesis. In this study, we have investigated CD68 and tryptase expression and their relationship with microvascular density (MVD) in chemo-resistant and chemosensitive patients affected by DLBCL. CD68 and tryptase expression as well as MVD were increased in chemo-resistant patients when compared with chemosensitive patients. Tryptase expression showed a positive correlation with MVD, supporting a role for mast cell in DLBCL tumor angiogenesis, while CD68 correlation with MVD was not significant, indicating a different role for TAMs than angiogenesis in DLBCL.

Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.

Mast cells (MCs) accumulate in the stroma surrounding tumors, where they secrete angiogenic cytokines and proteases, and an increased number of MCs have been demonstrated in angiogenesis associated with solid and hematological tumors. The aim of this study is to contribute to the knowledge of distribution of MCs in tumors, investigating the pattern of distribution of tryptase-positive MCs around the blood vessels in human endometrial carcinoma samples by introducing a quantitative approach to characterize their spatial distribution. The results have shown that in human endometrial cancer bioptic specimens the spatial distribution of MCs shows significant deviation from randomness as compared with control group in which, instead, the spatial distribution of MCs is consistent with a random distribution. These findings confirm that MCs enhance tumor angiogenesis and their preferential localization along blood vessels and sites of new vessel formation sustaining the suggestion for an association between MCs and angiogenesis. However, this spatial association between vessels and MCs might simply reflect migrating MCs from the blood stream at vessel growing sites.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

In this study we have investigated by real time polymerase chain reaction (RT-PCR) the expression of cytokines, which are well known angiogenic and anti-angiogenic molecules, during chick embryo development in four organs, namely the chorioallantoic membrane (CAM), heart, liver and optic tectum at four different stages. We have studied the expression of vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), hypoxia inducible factor-1 and -2 alpha (HIF-1α and HIF-2α), hepatocyte growth factor (HGF), and of endostatin. All the pro-angiogenic cytokines examined showed a progressive increase in their expression in brain, heart, and liver, through all the stages of development, and parallel endostatin reduces its expression. In contrast, in the CAM, all the pro-angiogenic factors examined, with the exception of ANG-1, showed a stably-decreased expression during development, whereas endostatin progressively increased its expression. The CAM as an extraembryonic membrane which mediates gas and nutrient exchange until hatching, may be considered an outer organ and not an intrinsic organ of the developing embryo in which the mechanisms regulating the development of the vascular tree are different.

Background Particulate matter (PM) is the most efficient vehicle for the inhalation and absorption of toxic substances into the body. Method The present study was aimed at testing the hypothesis that PM10 samples collected on quartz filters exert an angiogenic activity in vivo in the chick embryo chorioallantoic membrane (CAM) assay. Results When the low, medium, and high PM10 concentrations filters were tested in the CAM assay, an increasing number of microvessels was detectable after 4 days of applications of the filters. Moreover, at histological level, numerous microvessels and a dense inflammatory infiltrate were recognizable in the CAM mesenchyme. Conclusion Our data show a clear dose-response relationship between the dose variable (PM10 and Bap) and the outcome variable. So far, the PM10 target value is determined on the basis of regulatory agreements and is not health-based. In addition, the mere gravimetric measure of PM10 cannot be considered a fully reliable surrogate of the overall toxicity of the mixture.

Hyperbaric Oxygen Therapy (HBOT) is increasingly applied in different areas of medical practice. The oxy-hyperbarism effects are not well understood in cancer malignancy. One unique feature of cancer is the presence of hypoxic regions that are insensitive to conventional therapies. It is possible to alter the hypoxic state and produce reactive oxygen species for better treatment outcome by HBOT. In the present study, we determined the effects of HBOT on angiogenesis, a signature of cancer progression, by using the chick chorioallantoic membrane (CAM) in vivo assay. CAMs were exposed to 2.0 ATA (atmospheres absolute) for 30 min of hyperbaric oxygen on the 6(th) and 7(th) days of incubation (ED6, ED7). On the 10-11(th) day of incubation, CAMs were excised from eggs, fixed and analysed using APERIO ImageScope software. HBOT outcomes were evaluated quantifying the volumetric area occupied by blood vessels and calculating the number of blood vessel ramifications. Results indicated that CAMs treated at ED6 and ED7 had a significantly higher CAM vascularization and an increased number of blood vessel ramifications (+82% higher for ED6) compared to untreated CAMs (ED6=63.3PlusMinus;2.5 and ED7=57.7PlusMinus;5.5 vs. CTRL=34.7PlusMinus;2.5). Thus, HBOT induces an angiogenic response in treated CAMs through a classic sprouting mechanism.

Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.

The chick chorioallantoic membrane (CAM) assay is a well-established method to test the angiogenic stimulation or inhibition induced by molecules and cells administered onto the CAM. The quantification of blood vessels in the CAM assay relies on a semi-manual image analysis approach which can be time consuming when considering large experimental groups. Therefore we present here a simple and fast volumetric method to inspect differences in vascularization between experimental conditions related to the stimulation and inhibition of CAM angiogenesis based on the Positive Pixel Count algorithm embedded in the APERIO ImageScope software.

The spatial distribution of mast cells inside the tumor stroma has been little investigated. In this study, we have evaluated tumor mast cells distribution through the analysis of the morphological features of the spatial patterns generated by these cells, including size, shape, and architecture of the cell pattern. We have compared diffuse large B cells lymphoma (DLBCL) and systemic mastocytosis in two different anatomical localizations (lymph nodes for DLBCL and, respectively, bone marrow for mastocytosis). Results have indicated that, despite the high difference in size exhibited by the mast cells patterns in the two conditions, the spatial relationship between the mast cells forming the aggregates resulted similar, characterized by a significant tendency of the mast cells to self-organize in clusters.

Diffuse large B cell lymphoma (DLBCL) is recognized as the most common form of non-Hodgkin lymphoma (NHL), accounting for about 40 % of all cases of NHL. Among the cellular components of the tumor inflammatory infiltrate, T cells and mast cells have been demonstrated to be correlated with tumor angiogenesis. In this report, we have investigated CD3 and tryptase expression and their relationship with microvascular density (MVD) in DLBCL patients. Moreover, we determined the significance of CD3 expression in bulky and non-bulky disease. CD3 expression was significantly lower in bulky disease patients when compared to non-bulky ones. CD3 showed a positive correlation with tryptase and MVD, while multiple regression analysis efficaciously predicted MVD depending on CD3 and tryptase as predictors, supporting a complex interplay between these cells in sustaining tumor angiogenesis in DLBCL patients.

// Normann Steiner 1,2 , Domenico Ribatti 4,5 , Wolfgang Willenbacher 2,7 , Karin Johrer 3 , Johann Kern 1,7 , Christian Marinaccio 4 , Miguel Aracil 6 , Luis F. Garcia-Fernandez 6 , Guenther Gastl 2 , Gerold Untergasser 1,3,* and Eberhard Gunsilius 1,2,* 1 Laboratory for Tumor Biology & Angiogenesis, Innsbruck Medical University, Innsbruck, Austria 2 Department of Internal Medicine V, Innsbruck Medical University, Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria 4 Department of Basic Medical Sciences, Neurosciences, and Sensory Organs, University of Bari Medical School, Bari, Italy 5 National Cancer Institute “Giovanni Paolo II”, Bari, Italy 6 PharmaMar R&D, Colmenar Viejo, Madrid, Spain 7 Oncotyrol GmbH, Innsbruck, Austria * These authors contributed equally to this work Correspondence to: Eberhard Gunsilius, email: // Keywords : Marine drugs, Angiogenesis, Multiple Myeloma, CAM, Xenografts Received : December 02, 2014 Accepted : January 15, 2015 Published : January 31, 2015 Abstract Purpose: The prognosis of patients with multiple myeloma (MM) is still dismal despite recent improvements achieved by introducing new therapeutic agents. However, there remains an urgent need for progress in myeloma drug development. We here show that novel marine-derived compounds can exert potent anti-myeloma activity. Experimental Design: Nine marine-derived compounds were applied at low nM concentrations (0.1-100 nM) to MM cell lines (OPM-2, NCI-H929, U266, RPMI-8226), to primary human myeloma cells and to peripheral blood mononuclear cells. Apoptosis was determined by flow cytometry. In addition, eGFP-transgenic MM cell lines growing with mesenchymal cells from bone marrow were used to visualize tumors by fluorescence stereomicroscopy . Anti-myelomaactivities were studied in vitro in 3D spheroids and in vivo in myeloma xenografts on chicken embryos. Tumor size was analyzed by measuring GFP content with a GFP ELISA. Anti-angiogenic activities of compounds were tested in an in vivo gelatin sponge assay with conditioned media from primary bone marrow-derived endothelial cells. Results: We identified a subset of marine compounds with strong anti-myeloma activity in vitro and in vivo. Moreover, some of the compounds inhibited myeloma-related angiogenesis in the in vivo gelatin sponge assay. They merit further drug development to improve treatment options for MM.

Increased vascular density and endothelial cell proliferation have been demonstrated in multiple sclerosis (MS) white matter, as well as an elevated vascular endothelial growth factor expression was detected in reactive astrocytes of both active and inactive chronic demyelinated lesions and in sera of MS patients during clinical relapses. In this study, we have investigated the angiogenic activity of cerebrospinal fluid (CSF) samples from MS patients with different stages of disease by means of the chick embryo chorioallantoic membrane (CAM), a well-known assay to study angiogenesis in vivo. Results have shown that CSF samples from MS patients induced a significant (p < 0.05) angiogenic response in CAM in comparison with CSF from neurological controls. The vessel density was higher (p < 0.0001) in secondary (23.60 ± 1.14) and primary (23.50 ± 1.87) progressive patients in comparison with relapsing MS (17.25 ± 1.75) and clinically isolated syndrome suggestive of MS (13.00 ± 1.79), and a significant correlation (r = 0.611, p = 0.005) was found between the angiogenic response and disability level. The results of this preliminary report demonstrate for the first time an angiogenic activity in vivo of CSF samples from MS patients and confirm the importance of angiogenesis as a key event in MS pathogenesis and progression.

Angiogenesis is a hallmark of tumor growth and progression in solid and hematological malignancies. Different cellular components of the tumor microenvironment such as macrophages, mast cells, circulating endothelial cells and angiogenic factors, including vascular endothelial growth factor and its receptors are involved in the maintenance of Hodgkin Lymphoma. In this review article, we highlight relevant literature focusing on the relationships between angiogenesis and Hodgkin Lymphoma as well as discussing anti-angiogenic treatments in this malignancy.