Reconstructing tumor evolutionary histories and clone trees in polynomial-time with SubMARine

Tumors contain multiple subpopulations of genetically distinct cancer cells. Reconstructing their evolutionary history can improve our understanding of how cancers develop and respond to treatment. Subclonal reconstruction methods cluster mutations into groups that co-occur within the same subpopulations, estimate the frequency of cells belonging to each subpopulation, and infer the ancestral relationships among the subpopulations by constructing a clone tree. However, often multiple clone trees are consistent with the data and current methods do not efficiently capture this uncertainty; nor can these methods scale to clone trees with a large number of subclonal populations. Here, we formalize the notion of a partially-defined clone tree (partial clone tree for short) that defines a subset of the pairwise ancestral relationships in a clone tree, thereby implicitly representing the set of all clone trees that have these defined pairwise relationships. Also, we introduce a special partial clone tree, the Maximally-Constrained Ancestral Reconstruction (MAR), which summarizes all clone trees fitting the input data equally well. Finally, we extend commonly used clone tree validity conditions to apply to partial clone trees and describe SubMARine, a polynomial-time algorithm producing the subMAR, which approximates the MAR and guarantees that its defined relationships are a subset of those present in the MAR. We also extend SubMARine to work with subclonal copy number aberrations and define equivalence constraints for this purpose. Further, we extend SubMARine to permit noise in the estimates of the subclonal frequencies while retaining its validity conditions and guarantees. In contrast to other clone tree reconstruction methods, SubMARine runs in time and space that scale polynomially in the number of subclones. We show through extensive noise-free simulation, a large lung cancer dataset and a prostate cancer dataset that the subMAR equals the MAR in all cases where only a single clone tree exists and that it is a perfect match to the MAR in most of the other cases. Notably, SubMARine runs in less than 70 seconds on a single thread with less than one Gb of memory on all datasets presented in this paper, including ones with 50 nodes in a clone tree. On the real-world data, SubMARine almost perfectly recovers the previously reported trees and identifies minor errors made in the expert-driven reconstructions of those trees. The freely-available open-source code implementing SubMARine can be downloaded at https://github.com/morrislab/submarine.

Reconstructing tumor evolutionary histories and clone trees in polynomial-time with SubMARine

Tumors contain multiple subpopulations of genetically distinct cancer cells. Reconstructing their evolutionary history can improve our understanding of how cancers develop and respond to treatment. Subclonal reconstruction methods cluster mutations into groups that co-occur within the same subpopulations, estimate the frequency of cells belonging to each subpopulation, and infer the ancestral relationships among the subpopulations by constructing a clone tree. However, often multiple clone trees are consistent with the data and current methods do not efficiently capture this uncertainty; nor can these methods scale to clone trees with a large number of subclonal populations.Here, we formalize the notion of a partial clone tree that defines a subset of the pairwise ancestral relationships in a clone tree, thereby implicitly representing the set of all clone trees that have these defined pairwise relationships. Also, we introduce a special partial clone tree, the Maximally-Constrained Ancestral Reconstruction (MAR), which summarizes all clone trees fitting the input data equally well. Finally, we extend commonly used clone tree validity conditions to apply to partial clone trees and describe SubMARine, a polynomial-time algorithm producing the subMAR, which approximates the MAR and guarantees that its defined relationships are a subset of those present in the MAR. We also extend SubMARine to work with subclonal copy number aberrations and define equivalence constraints for this purpose. In contrast with other clone tree reconstruction methods, SubMARine runs in time and space that scales polynomially in the number of subclones.We show through extensive simulation and a large lung cancer dataset that the subMAR equals the MAR in > 99.9% of cases where only a single clone tree exists and that it is a perfect match to the MAR in most of the other cases. Notably, SubMARine runs in less than 70 seconds on a single thread with less than one Gb of memory on all datasets presented in this paper, including ones with 50 nodes in a clone tree.The freely-available open-source code implementing SubMARine can be downloaded at https://github.com/morrislab/submarine.Author summaryCancer cells accumulate mutations over time and consist of genetically distinct subpopulations. Their evolutionary history (as represented by tumor phylogenies) can be inferred from bulk cancer genome sequencing data. Current tumor phylogeny reconstruction methods have two main issues: they are slow, and they do not efficiently represent uncertainty in the reconstruction.To address these issues, we developed SubMARine, a fast algorithm that summarizes all valid phylogenies in an intuitive format. SubMARine solved all reconstruction problems in this manuscript in less than 70 seconds, orders of magnitude faster than other methods. These reconstruction problems included those with up to 50 subclones; problems that are too large for other algorithms to even attempt. SubMARine achieves these result because, unlike other algorithms, it performs its reconstruction by identifying an upper-bound on the solution set of trees. In the vast majority of cases, this upper bound is tight: when only a single solution exists, SubMARine converges to it > 99.9% of the time; when multiple solutions exist, our algorithm correctly recovers the uncertain relationships in more than 80% of cases.In addition to solving these two major challenges, we introduce some useful new concepts for and open research problems in the field of tumor phylogeny reconstruction. Specifically, we formalize the concept of a partial clone tree which provides a set of constraints on the solution set of clone trees; and provide a complete set of conditions under which a partial clone tree is valid. These conditions guarantee that all trees in the solution set satisfy the constraints implied by the partial clone tree.

Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation

The cytosolic (C) and mitochondrial (M) forms of phospho enolpyruvate carboxykinase (PEPCK; EC 4.1.1.32 ) are encoded by two different nuclear genes in mouse, human, and chicken. Our objective was to clone the two forms of PEPCK for bovine and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2,592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449-nt partial clone of the 3′ end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCK-M. The coding sequence of bovine PEPCK-C and coding sequence of the partial PEPCK-M clone were 58% similar but the similarities in the 3′-untranslated regions were negligible. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression, but expression of PEPCK-C mRNA was transiently increased during early lactation, indicating that enhanced hepatic gluconeogenesis during this period may be tied to enhanced capacity for cytosolic rather than mitochondrial formation of phosphoenolpyruvate.

Mimicry between neurokinin-1 and fibronectin may explain the transport and stability of increased substance P immunoreactivity in patients with bone marrow fibrosis

Bone marrow (BM) fibrosis may occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases. In this study, the role of substance P (SP), a peptide with pleiotropic functions, was examined. Some of its functions—angiogenesis, fibroblast proliferation, and stimulation of BM progenitors—are amenable to inducing BM fibrosis. Indeed, a significant increase was found in SP-immunoreactivity (SP-IR) in the sera of patients with BM fibrosis (n = 44) compared with the sera of patients with hematologic disorders and no histologic evidence of fibrosis (n = 46) (140 ±12 vs 18 ±3; P < .01). Immunoprecipitation of sera SP indicated that this peptide exists in the form of a complex with other molecule(s). It was, therefore, hypothesized that SP might be complexed with NK-1, its natural receptor, or with a molecule homologous to NK-1. To address this, 3 cDNA libraries were screened that were constructed from pooled BM stroma or mononuclear cells with an NK-1 cDNA probe. A partial clone (clone 1) was retrieved that was 97% homologous to the ED-A region of fibronectin (FN). Furthermore, sequence analyses indicated that clone 1 shared significant homology with exon 5 of NK-1. Immunoprecipitation and Western blot analysis indicated co-migration of SP and FN in 27 of 31 patients with BM fibrosis. Computer-assisted molecular modeling suggested that similar secondary structural features between FN and NK-1 and the relative electrostatic charge might explain a complex formed between FN (negative) and SP (positive). This study suggests that SP may be implicated in the pathophysiology of myelofibrosis, though its role would have to be substantiated in future research.

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

Although Na+-K+-2Cl− cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [3H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s−1. Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na+-K+-2Cl− cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.