To a Mouse, by Robert Burns

Little, cunning, cowering, timorous beast,
Oh, what a panic is in your breast!
You need not start away so hasty
With bickering prattle!
I would be loath to run and chase you,
With murdering paddle!

I'm truly sorry man's dominion
Has broken Nature's social union,
And justifies that ill opinion
Which makes you startle
At me, your poor, earth-born companion
And fellow mortal!

I doubt not, sometimes, that you may steal;
What then? Poor beast, you must live!
An odd ear in twenty-four sheaves
Is a small request;
I will get a blessing with what is left,
And never miss it.

Your small house, too, in ruin!
Its feeble walls the winds are scattering!
And nothing now, to build a new one,
Of coarse green foliage!
And bleak December's winds ensuing,
Both bitter and piercing!

You saw the fields laid bare and empty,
And weary winter coming fast,
And cozy here, beneath the blast,
You thought to dwell,
Till crash! The cruel plough passed
Out through your cell.

That small heap of leaves and stubble,
Has cost you many a weary nibble!
Now you are turned out, for all your trouble,
Without house or holding,
To endure the winter's sleety dribble,
And hoar-frost cold.

But Mouse, you are not alone,
In proving foresight may be vain:
The best-laid schemes of mice and men
Go often askew,
And leave us nothing but grief and pain,
For promised joy!

Still you are blessed, compared with me!
The present only touches you:
But oh! I backward cast my eye,
On prospects dreary!
And forward, though I cannot see,
I guess and fear!

By Kevin E. Noonan —

Measured by global dispersal alone, the common house mouse (Mus musculus ssp.) is the most successful invasive mammalian species.  Perhaps surprisingly, the origin and history of this dispersal in the Western world has not be fully elucidated, but it is clear that mice have dispersed throughout the world in concert with human migration (posing a dual threat to native species diversity).

That situation has changed, in a report by an international cadre of researchers reported in Nature last month entitled "Tracking the Near Eastern origins and European dispersal of the western house mouse."  These researchers assessed 829 specimens from 43 archeological sites from Southwestern Asia and Southeastern Europe representing from 40,000 to 3,000 years before the present day, performing mitochondrial genetic analyses on 85 samples and combining these results with radiocarbon dating and geometric morphometrics numerical taxonomy.

It has been known heretofore that M. musculus arose in the Indian subcontinent and differentiated during the Pleistocene into the three surviving mouse subspecies (M. m. domesticus, M. m. musculus, and M. m. castaneus).  The researchers report that the commensal behavior of the house mouse arose in the Levant about 14,500 years ago with hunter-gatherers in that region developing a more sedentary civilization (and they speculate that this promoted domestication of cats because those civilizations did not share Burns' sympathy for murine depredations of perhaps scarce proto-agricultural provisions).  Mouse coexistence with man was promoted by the emergence of human agriculture and agricultural settlements ~12,000 years ago and the invasion of Europe beginning with "stowaway" transmission to Cyprus about 10,800 years ago (followed by evidence of human introduction of cats to the island nation around 9,500 years ago).  The results of the historical researches reported here indicate that frank colonization of the European mainland began about 6,500 years ago in Eastern Europe and 4,000 years ago in Southwestern Europe with the development of "proto-urbanism" and trade (the authors again speculating that these developments also fostered human-facilitated dispersal of cats in Europe).

These researchers presented data from "five key chronological phases of human history in the studied area: (1) the pre-sedentism period: 40,000–15,500 cal BP, (2) the early sedentary communities of hunter-gatherers: 15,500–12,000 cal BP, (3) the early agrarian economy and dispersal in the Near East and Cyprus: 12,000–8,500 cal BP, (4) the Neolithic dispersal towards Europe: 8,500–6,500 cal BP, and (5) the Late Neolithic/Bronze Age exchange and trade networks: 6,500–3,000 cal BP."

The earliest fossil evidence was obtained from the Iranian Plateau from Middle and Paleolithic cave deposits left from birds of prey.  The earliest comingling of human and mouse remains were found around the Pleistocene-Holocene transition in sedentary open-air settlements of hunter-gatherers in both the Northern and Southern Levant.  Later M. musculus remains found in association with human settlements were found in "Pre-Pottery Neolithic (PPN)"farming villages in Southwest Asia and in Cyprus.  Mice were also found in association with human settlements in Transcaucasia between 5,000 and 4,000 BP.  These researchers found no spread of mice into the Eastern Mediterranean and Southeastern Europe beyond Cyprus during the Neolithic, with mice found in Greece from those times being the wild M. macedonicus species.  M. musculus invasion in the Aegean was delayed until the Bronze Age in Crete and Santorini, which the authors say suggests dispersal with humans through maritime networks (confirmed somewhat by a finding of a mouse mandible in the cargo of a shipwreck of the south shore of Asia Minor).  Again, somewhat surprisingly, the authors report that M. musculus musculus, rather than M. musculus domesticus, was the first subspecies to colonize Europe in association with human migration.

Nevertheless, the pattern of human-mouse association in the Levant and Asia Minor during the Neolithic showed M. musculus domesticus in early human proto-urban context.  However the pattern of the evidence showed that it was "the impact of sedentism on ecosystems and the ecology of organisms (i.e. reduction of predation and competition pressures, climatic buffer etc.) [that] was the catalyst for the commensal relationship between mice and humans rather than the emergence of agriculture systems with large-scale grain storage" (something that arose two thousand years later than these Neolithic settlements).  In addition, M. musculus domesticus was limited to larger settlements, with M. macedonicus being found in smaller human settlements.  This is not to say that the emergence of human agriculture did not play a major role in the development of human-mouse commensalism; as the authors state, "[t]he occurrence of M. m. domesticus in all the [Neolithic settlements studied] suggests that the emergence of the agricultural system was the key driving force in the house mouse's commensal trajectory" and correlates with "plant cultivation of wild cereals and pulses [and] the emergence of the first settlement with communal buildings and cereal storage."  The advantages (for the mice) included "greater protection against predators and competitors, a buffer from temperature fluctuation, and a constant food supply due to large scale grain storage" from the PPN and, as a consequence, the house mouse became an "anthro-dependent organism."

Regarding the lack of evidence that M. musculus domesticus followed the "mainland-island" pattern of invasion, the authors speculate that "neither the ecological niche nor the migrant flow could sustain this biological invasion model" because during the Neolithic settlements in mainland Greece and the Balkans were small settlements lacking communal food storage facilities.  In addition, indigenous M. macedonicus and M. spicilegus populations provided a competitive barrier to M. musculus domesticus invasion.  This changed "[o]nly [with] the intensification of maritime trade with the Near East driven by Bronze Age cities and the increasing size and stability of settlements associated with this migrant flow."  But the authors admit that the exact route by which M. musculus musculus invaded Europe is still not firmly established and possible routes include from the Transcaucusus and the western shores of the Black Sea and the Bosphorus.

Finally, the paper considers the synergy between house mouse commensalism with human settlements and domestication of cats.  Formerly the locus (both temporal and spatial) of human cat domestication was the Nile Valley around 6,000 years ago.  Current information, including in this paper, is that humans domesticated cats in association with Neolithic settlements in the Levant and later proto-cities in Cyprus, with Felis sylvestris being introduced to the island about 11,000 years ago, which correlates with the earliest cereal cultivation and the earliest evidence of M. musculus domesticus in the island.  This pattern of cat introduction in response to mouse infestation of human settlements is supported by the presence of cats in Greece and the Balkans in the same timeframe (~6000 years ago) as the house mouse, the authors stating:

We propose as a hypothesis that the earliest cat dispersal towards Europe was driven by M. m. musculus biological invasion during the Late Neolithic/Chalcolithic, when the size of the proto-urban settlements and the catchment for grain production generated rodent pests and therefore the need to tackle them with cat predation.  The later dispersal in continental Greece on the other hand could have been pushed by the later M. m. domesticus dispersal associated with the development of Bronze Age urbanisation and the need for pest control in the Balkan peninsula.

The authors concede, "an extensive survey for cat remains associated with direct radiocarbon dating and high-throughput paleogenetics analyses to capture recent phylogeographic lineages within F.s.lybica needs to be pursued."  These include the following proposed (and likely currently pursued) studies, the prospects of which provide the conclusion of the paper:

The models of origin and dispersal for the two house mouse sub-species in Southwestern Asia and Europe generated by this study need to be further tested using high-throughput sequencing and paleogenomic approaches.  Retrieving mitochondrial haplotypes from ancient house mouse will produce dated phylogeographic inferences about the colonization history of both sub-species in Europe and indirect clues about past human dispersal and trading networks.  Genome-wide studies of ancient commensal populations across Southwestern Asia and along a time series from the Late Glacial to the Iron Age will potentially allow the investigation of (1) the genetic signatures for the behavioural selection involved in the commensalism process, (2) specific phenotypic traits separating M. musculus sub-species from other wild species dwelling around the Mediterranean, such as the tail length, longer in M. musculus sub-species and (3) the amount of genetic isolation and introgression with autochtonous Mus species involved in the evolutionary process of the house mouse.

* Thomas Cucchi, Katerina Papayianni, Sophie Cersoy, Laetitia Aznar-Cormano, Antoine Zazzo, Régis Debruyne, Rémi Berthon, Adrian Bălășescu, Alan Simmons, François Valla, Yannis Hamilakis, Fanis Mavridis, Marjan Mashkour, Jamshid Darvish, Roohollah Siahsarvi, Fereidoun Biglari, Cameron A. Petrie, Lloyd Weeks, Alireza Sardari, Sepideh Maziar, Christiane Denys, David Orton, Emma Jenkins, Melinda Zeder, Jeremy B. Searle, Greger Larson, François Bonhomme, Jean-Christophe Auffray & Jean-Denis Vigne

By Kevin E. Noonan —

The inherent, ineluctable unpredictability of biology can be the basis for biological patent claims being non-obvious (lacking the requisite "reasonable expectation of success"; see, e.g., OSI Pharmaceuticals v. Apotex) and for the greater quantum of disclosure necessary to satisfy the written description and enablement requirements of § 112 (see, e.g., Amgen v. Sanofi), despite complaints from the life sciences patent bar that these increased requirements are improper doctrinally and unfair.  These two different characteristics can be frequently in tension for patenting in the life sciences, it being difficult to maintain on the one hand that there is insufficient expectation of success for a claim to be obvious and on the other hand that deficiencies in disclosure can be appropriately supplemented by the knowledge of one of ordinary skill.

This unpredictability was illustrated in a paper recently published in Nature Medicine, entitled "Common germline variants of the human APOE gene modulate melanoma progression and survival."  These authors* showed (somewhat paradoxically) that one variant of the human APOE gene (APOE2) was associated with a propensity for tumor cells to metastasize, while a different variant (APOE4), which has been known for several years to be associated with development of Alzheimer's disease (see Strittmatter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 1977-81), exhibited a metastasis-inhibiting effect (and APOE2 itself can have a protective effect on development of late-onset Alzheimer's; see Corder et al., 1994, Nat. Genet. 7: 180-84).

The experiments were performed in mice expressing human APOE4 or APOE2 by genetic replacement of the mouse analogs.  Differences in melanoma tumor growth in these two mouse strains carrying these different human APOE genes were shown by comparing mouse melanoma tumor growth as shown in this Figure:

(where YUMM3.3 and YUMMER1.7 are murine melanoma cell lines).

The protective effects against metastasis of APOE4 were compared with APOE2 in these mice was demonstrated by tail vein injection of B16F10 melanoma cells, an established metastasis animal model.  The human APOE4-bearing mice had a phenotype of enhanced anti-tumor immune response and improved outcomes under PD1 immune checkpoint blockade.

Because APOE was known to have modulatory effects on immune response, flow cytometric analysis of APOE gene variants in mice was performed and showed "enhanced recruitment of CD45+ leukocytes in animals bearing various melanoma tumors in APOE4 mice compared with APOE2 mice."  Proportions of immune suppressor cells (Ly6G+ granulocytic myeloid-derived suppressor cells) were found to be diminished in APOE4-bearing mice, while these mice showed increased proportions of antitumor effector cells such as natural killer (NK) cells and CD8+ T cells.  These results were confirmed by single-cell RNA sequencing for detecting lineage-specific gene expression.  Further experiments showed that T cell depletion "completely abrogated" differences in melanoma tumor growth between human APOE4– and human APOE2-bearing mice.  The authors concluded that "[t]hese data suggest that APOE genotype modulated both the abundance and the functional state of the tumor immune microenvironment, with the APOE4 variant eliciting an enhanced anti-tumor immune profile relative to the APOE2 variant."  These authors also showed that APOE4 suppressed melanoma cell invasion and endothelial recruitment (involved in angiogenesis), which was consistent with lower blood vessel density in APOE4 mice.

In addition to these mouse studies, the authors assessed APOE genotype association in melanoma human patients from The Cancer Genome Atlas (TCGA).  Neither of these APOE variants was enriched in the database, which the authors said indicated neither gene was involved in increased melanoma incidence.  However, APEO4 carriers had improved survival, with 10.1 years for these patients versus 2.1 years for APEO2 carriers.  This outcome was surprising due to the reduced longevity associated with APOE4 carriers, which the authors attributed to the high rates of melanoma-associated death.  These results demonstrated that "germline genetic variants of APOE differentially associated with survival in patients with advanced melanoma who were at increased risk for melanoma-associated death and metastasis."

PD-1 immunotherapy is a commonly used treatment for melanoma, and "APOE4 mice survived significantly longer than APOE2 mice upon anti-PD1 treatment, suggesting that APOE genotype modulates melanoma outcome also in the context of immunotherapy," according to the results shown in the paper.  In humans,  "APOE4 and APOE2 carriers exhibited the longest and shortest survival outcomes, respectively, upon anti-PD1 therapy," consistent with the results in mice.

The final set of experimental results reported in this paper involved pharmacologic activation of APOE through liver X receptors, which are "nuclear hormone receptors that transcriptionally activate several genes implicated in cholesterol and lipid metabolism, including APOE."  In mice, this effect was completely abrogated in APOE2 mice but showed "robust anti-tumor effects" upon treatment in APOE4 mice.  The authors concluded from these results that "distinct APOE genotypes elicited differential responsiveness to LXR agonistic therapy and might serve as potential genetic biomarkers for current clinical efforts investigating the use of LXR agonism in cancer therapy."

The authors provide the following context for the results set forth in their paper:

Our findings have several potential clinical implications.  Most importantly, they suggest that common germline variants might serve as biomarkers to identify patients with melanoma who are at high risk for metastatic relapse and melanoma-associated death for treatment with adjuvant systemic therapy.  Notably, these clinical association findings will need to be assessed in prospective studies.  It will be important to also assess the effect of APOE genotype on the outcome of additional cancer types.  More generally, our findings support the notion that hereditary germline variants in the same gene can positively or negatively affect future progression and survival outcomes and responsiveness to therapy in a common human malignancy.

Authors: Benjamin N. Ostendorf^a, Jana Bilanovic^a,  Nneoma Adaku^a, Kimia N. Tafreshian^a, Bernardo Tavora^a, Roger D. Vaughan^b & Sohail F. Tavazoie^a 

^a Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA
^b Department of Biostatistics, The Rockefeller University, New York, NY, USA