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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.