The bone marrow microenvironment at single-cell resolution
Tikhonova, Anastasia N; Dolgalev, Igor; Hu, Hai; Sivaraj, Kishor K; Hoxha, Edlira; Cuesta-DomÃnguez, Ãlvaro; Pinho, Sandra; Akhmetzyanova, Ilseyar; Gao, Jie; Witkowski, Matthew; Guillamot, Maria; Gutkin, Michael C; Zhang, Yutong; Marier, Christian; Diefenbach, Catherine; Kousteni, Stavroula; Heguy, Adriana; Zhong, Hua; Fooksman, David R; Butler, Jason M; Economides, Aris; Frenette, Paul S; Adams, Ralf H; Satija, Rahul; Tsirigos, Aristotelis; Aifantis, Iannis
The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.
Extensive Remodeling of the Immune Microenvironment in B Cell Acute Lymphoblastic Leukemia
Witkowski, Matthew T; Dolgalev, Igor; Evensen, Nikki A; Ma, Chao; Chambers, Tiffany; Roberts, Kathryn G; Sreeram, Sheetal; Dai, Yuling; Tikhonova, Anastasia N; Lasry, Audrey; Qu, Chunxu; Pei, Deqing; Cheng, Cheng; Robbins, Gabriel A; Pierro, Joanna; Selvaraj, Shanmugapriya; Mezzano, Valeria; Daves, Marla; Lupo, Philip J; Scheurer, Michael E; Loomis, Cynthia A; Mullighan, Charles G; Chen, Weiqiang; Rabin, Karen R; Tsirigos, Aristotelis; Carroll, William L; Aifantis, Iannis
A subset of B cell acute lymphoblastic leukemia (B-ALL) patients will relapse and succumb to therapy-resistant disease. The bone marrow microenvironment may support B-ALL progression and treatment evasion. Utilizing single-cell approaches, we demonstrate B-ALL bone marrow immune microenvironment remodeling upon disease initiation and subsequent re-emergence during conventional chemotherapy. We uncover a role for non-classical monocytes in B-ALL survival, and demonstrate monocyte abundance at B-ALL diagnosis is predictive of pediatric and adult B-ALL patient survival. We show that human B-ALL blasts alter a vascularized microenvironment promoting monocytic differentiation, while depleting leukemia-associated monocytes in B-ALL animal models prolongs disease remission inÂ vivo. Our profiling of the B-ALL immune microenvironment identifies extrinsic regulators of B-ALL survival supporting new immune-based therapeutic approaches for high-risk B-ALL treatment.
Connecting the Dots: Resolving the Bone Marrow Niche Heterogeneity
Dolgalev, Igor; Tikhonova, Anastasia N
Single-cell sequencing approaches have transformed our understanding of stem cell systems, including hematopoiesis and its niche within the bone marrow. Recent reports examined the bone marrow microenvironment at single-cell resolution at steady state, following chemotherapy treatment, leukemic onset, and aging. These rapid advancements significantly informed our understanding of bone marrow niche heterogeneity. However, inconsistent representation and nomenclature among the studies hinder a comprehensive interpretation of this body of work. Here, we review recent reports interrogating bone marrow niche architecture and present an integrated overview of the published datasets.
Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression
Cimmino, Luisa; Dolgalev, Igor; Wang, Yubao; Yoshimi, Akihide; Martin, Gaelle H; Wang, Jingjing; Ng, Victor; Xia, Bo; Witkowski, Matthew T; Mitchell-Flack, Marisa; Grillo, Isabella; Bakogianni, Sofia; Ndiaye-Lobry, Delphine; Martin, Miguel Torres; Guillamot, Maria; Banh, Robert S; Xu, Mingjiang; Figueroa, Maria E; Dickins, Ross A; Abdel-Wahab, Omar; Park, Christopher Y; Tsirigos, Aristotelis; Neel, Benjamin G; Aifantis, Iannis
Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and alpha-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.
Integrative genomic profiling of human prostate cancer
Taylor, Barry S; Schultz, Nikolaus; Hieronymus, Haley; Gopalan, Anuradha; Xiao, Yonghong; Carver, Brett S; Arora, Vivek K; Kaushik, Poorvi; Cerami, Ethan; Reva, Boris; Antipin, Yevgeniy; Mitsiades, Nicholas; Landers, Thomas; Dolgalev, Igor; Major, John E; Wilson, Manda; Socci, Nicholas D; Lash, Alex E; Heguy, Adriana; Eastham, James A; Scher, Howard I; Reuter, Victor E; Scardino, Peter T; Sander, Chris; Sawyers, Charles L; Gerald, William L
Annotation of prostate cancer genomes provides a foundation for discoveries that can impact disease understanding and treatment. Concordant assessment of DNA copy number, mRNA expression, and focused exon resequencing in 218 prostate cancer tumors identified the nuclear receptor coactivator NCOA2 as an oncogene in approximately 11% of tumors. Additionally, the androgen-driven TMPRSS2-ERG fusion was associated with a previously unrecognized, prostate-specific deletion at chromosome 3p14 that implicates FOXP1, RYBP, and SHQ1 as potential cooperative tumor suppressors. DNA copy-number data from primary tumors revealed that copy-number alterations robustly define clusters of low- and high-risk disease beyond that achieved by Gleason score. The genomic and clinical outcome data from these patients are now made available as a public resource.
Prognostic relevance of integrated genetic profiling in acute myeloid leukemia
Patel, Jay P; Gonen, Mithat; Figueroa, Maria E; Fernandez, Hugo; Sun, Zhuoxin; Racevskis, Janis; Van Vlierberghe, Pieter; Dolgalev, Igor; Thomas, Sabrena; Aminova, Olga; Huberman, Kety; Cheng, Janice; Viale, Agnes; Socci, Nicholas D; Heguy, Adriana; Cherry, Athena; Vance, Gail; Higgins, Rodney R; Ketterling, Rhett P; Gallagher, Robert E; Litzow, Mark; van den Brink, Marcel R M; Lazarus, Hillard M; Rowe, Jacob M; Luger, Selina; Ferrando, Adolfo; Paietta, Elisabeth; Tallman, Martin S; Melnick, Ari; Abdel-Wahab, Omar; Levine, Ross L
BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. METHODS: We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients. RESULTS: We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). CONCLUSIONS: We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).
Mutations in GNA11 in uveal melanoma
Van Raamsdonk, Catherine D; Griewank, Klaus G; Crosby, Michelle B; Garrido, Maria C; Vemula, Swapna; Wiesner, Thomas; Obenauf, Anna C; Wackernagel, Werner; Green, Gary; Bouvier, Nancy; Sozen, M Mert; Baimukanova, Gail; Roy, Ritu; Heguy, Adriana; Dolgalev, Igor; Khanin, Raya; Busam, Klaus; Speicher, Michael R; O'Brien, Joan; Bastian, Boris C
BACKGROUND: Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. METHODS: We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. RESULTS: We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. CONCLUSIONS: Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).
Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies
Abdel-Wahab, Omar; Mullally, Ann; Hedvat, Cyrus; Garcia-Manero, Guillermo; Patel, Jay; Wadleigh, Martha; Malinge, Sebastien; Yao, JinJuan; Kilpivaara, Outi; Bhat, Rukhmi; Huberman, Kety; Thomas, Sabrena; Dolgalev, Igor; Heguy, Adriana; Paietta, Elisabeth; Le Beau, Michelle M; Beran, Miloslav; Tallman, Martin S; Ebert, Benjamin L; Kantarjian, Hagop M; Stone, Richard M; Gilliland, D Gary; Crispino, John D; Levine, Ross L
Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.
Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition
Wei, Wei; Geer, Mitchell J; Guo, Xinyi; Dolgalev, Igor; Sanjana, Neville E; Neel, Benjamin G
SHP2 (PTPN11) acts upstream of SOS1/2 to enable RAS activation. Allosteric SHP2 inhibitors (SHP2i) in the clinic prevent SHP2 activation, block proliferation of RTK- or cycling RAS mutant-driven cancers, and overcome "adaptive resistance." To identify SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive cell lines, recovering genes expected to cause resistance (NF1, PTEN, CDKN1B, LZTR1, and RASA2) and novel targets (INPPL1, MAP4K5, epigenetic modifiers). We screened 14 additional lines with a focused CRISPR library targeting common "hits" from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2/STK40 (6/14), and INPPL1 (5/14). INPPL1, MAP4K5, or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not lipid phosphatase activity. MAP4K5 acted upstream of MEK through a kinase-dependent target(s); LZTR1 had cell-dependent effects on RIT and RAS stability. INPPL1, MAP4K5, or LZTR1 deletion also conferred SHP2i resistance in vivo. Defining the SHP2i resistance landscape could suggest effective combination approaches.
An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma
Davalos, Veronica; Lovell, Claudia D; Von Itter, Richard; Dolgalev, Igor; Agrawal, Praveen; Baptiste, Gillian; Kahler, David J; Sokolova, Elena; Moran, Sebastian; Piqué, Laia; Vega-Saenz de Miera, Eleazar; Fontanals-Cirera, Barbara; Karz, Alcida; Tsirigos, Aristotelis; Yun, Chi; Darvishian, Farbod; Etchevers, Heather C; Osman, Iman; Esteller, Manel; Schober, Markus; Hernando, Eva
Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.