Addiction to multiple oncogenes can be exploited to prevent the emergence of therapeutic resistance

Many cancers exhibit sensitivity to the inhibition of a single genetic lesion, a property that has been successfully exploited with oncogene-targeted therapeutics. However, inhibition of single oncogenes often fails to result in sustained tumor regression due to the emergence of therapy-resistant cells. Here, we report that MYC-driven lymphomas frequently acquire activating mutations in β-catenin, including a previously unreported mutation in a splice acceptor site. Tumors with these genetic lesions are highly dependent on β-catenin for their survival and the suppression of β-catenin resulted in marked apoptosis causally related to a decrease in Bcl-xL expression. Using a novel inducible inhibitor of β-catenin, we illustrate that, although MYC withdrawal or β-catenin inhibition alone results in initial tumor regression, most tumors ultimately recurred, mimicking the clinical response to single-agent targeted therapy. Importantly, the simultaneous combined inhibition of both MYC and β-catenin promoted more rapid tumor regression and successfully prevented tumor recurrence. Hence, we demonstrated that MYC-induced tumors are addicted to mutant β-catenin, and the combined inactivation of MYC and β-catenin induces sustained tumor regression. Our results provide a proof of principle that targeting multiple oncogene addicted pathways can prevent therapeutic resistance.Cancer cells are highly sensitive to the targeted inhibition of single driver mutations, eliciting a phenomenon known as “oncogene addiction” (1). The identification of genetic dependencies in multiple tumor types has resulted in the development of several molecularly targeted therapeutics, including the BCR-ABL kinase inhibitor imatinib for the treatment of chronic myelogenous leukemia (CML), the EGFR kinase inhibitor gefitinib for the treatment of non–small cell lung cancer (NSCLC), and the BRAF kinase inhibitor vemurafenib for the treatment of advanced melanoma (2–4). Although these oncogene-targeted agents have provided promising clinical responses, many patients ultimately experience a recurrence of their disease due to the development of drug resistance (4–6). Thus, it has become evident that monotherapy with targeted drugs is insufficient for achieving sustained tumor regression.Resistance to targeted therapy can arise through multiple mechanisms, depending on the tumor type and the targeted oncogenic pathway (7). Cells frequently acquire resistance through mutations in the targeted oncogene itself that disrupt drug binding, as in the case of BCR-ABL and EGFR (8, 5, 6). In addition, resistance to EGFR inhibition in NSCLC and BRAF inhibition in melanoma has been found to occur through a variety of mechanisms that activate downstream signaling proteins or alternative pathways, which can functionally substitute for loss in activity of the targeted oncogene (9–11). Although significant progress has been made in the identification and inhibition of resistance pathways, it may prove challenging to anticipate and suppress all of the potential mechanisms of resistance for each oncogene-addicted cancer and targeted therapeutic agent.Combination therapy has been successfully applied to prevent resistance in the treatment of infectious diseases such as HIV (12, 13) and tuberculosis (14). In the context of oncogene-targeted therapy for cancer, it has been proposed that a similar strategy, using combinations directed against multiple dependencies, is the most likely to prevent resistance (7). Indeed, mathematical modeling indicates that targeting at least two independently required pathways may be sufficient to prevent tumor recurrence (15). However, there exists little experimental evidence directly testing such an approach and it remains unclear which combinations of targets would be most effective at inducing long-term remissions.MYC is one of the most frequently amplified oncogenes in human cancer (16). In the Eμ-tTA/tetO-MYC conditional mouse model, overexpression of MYC results in the development of aggressive T-cell lymphoma, and MYC inactivation in established tumors is sufficient to induce tumor regression through processes such as proliferative arrest, cellular senescence, apoptosis, and the shutdown of angiogenesis (17–19). The extent of regression is dependent on both cell-intrinsic and host-dependent contexts, and in particular, tumors frequently recur following MYC inactivation in the absence of an intact adaptive immune system (20). Recurring tumors restore expression of the MYC transgene or up-regulate expression of endogenous Myc, demonstrating that resistance occurs primarily through reactivation of the MYC pathway (21). Thus, MYC oncogene addiction and tumor recurrence in the Eμ-tTA/tetO-MYC lymphoma model resembles the clinical course of human cancers treated with single agent targeted therapy.Here, we demonstrate that the combined inactivation of two oncogene addiction pathways can result in sustained tumor regression. Moreover, we describe a previously unidentified splice acceptor site mutation in β-catenin that is associated with MYC-induced lymphomagenesis. Tumors with mutations in β-catenin are also highly addicted to this mutant gene product for their survival. We demonstrate that in MYC-induced lymphomas, combined addiction to both MYC and β-catenin can be exploited in a rational manner to prevent the emergence of therapeutic resistance.     

Flow disturbances generated by feeding and swimming zooplankton

Interactions between planktonic organisms, such as detection of prey, predators, and mates, are often mediated by fluid signals. Consequently, many plankton predators perceive their prey from the fluid disturbances that it generates when it feeds and swims. Zooplankton should therefore seek to minimize the fluid disturbance that they produce. By means of particle image velocimetry, we describe the fluid disturbances produced by feeding and swimming in zooplankton with diverse propulsion mechanisms and ranging from 10-µm flagellates to greater than millimeter-sized copepods. We show that zooplankton, in which feeding and swimming are separate processes, produce flow disturbances during swimming with a much faster spatial attenuation (velocity u varies with distance r as u ∝ r⁻³ to r⁻⁴) than that produced by zooplankton for which feeding and propulsion are the same process (u ∝ r⁻¹ to r⁻²). As a result, the spatial extension of the fluid disturbance produced by swimmers is an order of magnitude smaller than that produced by feeders at similar Reynolds numbers. The “quiet” propulsion of swimmers is achieved either through swimming erratically by short-lasting power strokes, generating viscous vortex rings, or by “breast-stroke swimming.” Both produce rapidly attenuating flows. The more “noisy” swimming of those that are constrained by a need to simultaneously feed is due to constantly beating flagella or appendages that are positioned either anteriorly or posteriorly on the (cell) body. These patterns transcend differences in size and taxonomy and have thus evolved multiple times, suggesting a strong selective pressure to minimize predation risk.Zooplankters move to feed, find food, and find mates, so moving is critical to the efficient execution of essential functions. However, moving comes at a predation risk: Swimming increases the predator encounter velocity (encounter rate increases with prey velocity to a power ≤1), and feeding and swimming generate fluid disturbances that may be perceived by rheotactic predators, thus increasing the predator’s detection distance (encounter rate increases with detection distance squared) (1–5). So, the advantages of moving and feeding must be traded off against the associated risks, and organisms should aim at moving and foraging in ways that reduce the predation risk and optimize the trade-off (6, 7). They may do so by moving in patterns that minimize encounter rates (8) and/or they may feed and propel themselves in ways that generate only small fluid disturbances (9). For example, theoretical models suggest that zooplankton that swim by a sequence of jumps may create a smaller fluid disturbance than similar-sized ones that swim smoothly (9), that a hovering zooplankter generates a larger fluid signal than one that cruises through the water (10, 11), and that a zooplankter moving at low Reynolds numbers will generate a relatively larger fluid signal than one moving at higher Reynolds numbers (11). Thus, motility patterns and propulsion modes may strongly influence predation risk and must be subject to strong selection pressure during evolution.Zooplankton span a huge taxonomic diversity and a large size range (from microns to centimeters) and their propulsion mechanisms vary substantially (12). Unicellular plankton may use one or more flagella or cilia, and the flagella may be smooth or plumose, which has implications for whether the cell is pulled or pushed by the beating flagellum (13). Ciliates may have the cilia rather evenly distributed on the cell surface or concentrated on certain parts of the cell, typically either anteriorly or as an equatorial band. Small animals may have an anterior “corona” of cilia (e.g., rotifers and many pelagic invertebrate larvae) to generate feeding currents and propulsion, or they may have beating or vibrating appendages that can be positioned anteriorly, ventrally, or laterally. The implications and potential adaptive value of this diversity of propulsion modes for feeding and survival are largely unexplored.Various idealized models, simplifying the swimming organisms to combinations of point forces acting on the water, have been used to describe the fluid disturbance generated by moving and feeding plankton. A self-propelled plankton is often described by a so-called stresslet (two oppositely directed point forces of equal magnitude), a hovering one by a stokeslet (a stationary point force), and a jumping animal by an impulsive stresslet (a stresslet working impulsively) (9, 11, 12). These highly idealized models yield very different predictions of the spatial attenuation of the fluid disturbance and, thus, of how far away the feeding and swimming animal can be detected. A few studies have compared observed flow patterns with those predicted from these simple models and in some cases found fair comparisons (4, 14–17). However, numerical simulations as well as observations of self-propelled microplankton have demonstrated that the distribution of propulsion forces, i.e., the position of flagella, cilia, or appendages on the (cell) body, may have a profound effect on the imposed fluid flow (18, 19). Also, most of the idealized models ignore the fact that swimming in most cases is unsteady, which leads to fluctuating flows at scales smaller than the Stokes length scale (ν/ω, where ν is the kinematic viscosity and ω is the beat frequency) (e.g., ref. 19). The simple, idealized models hitherto applied may be insufficient to represent the diverse propulsion modes observed in real organisms and to understand the associated trade-offs.Feeding and swimming are often part of the same process in zooplankton. Many zooplankton generate a feeding current that at the same time propels the animal through the water. In others, feeding and swimming are separate processes. For example, ambush feeding “sit-and-wait” zooplankters do not move as part of feeding but may swim to undertake vertical migration or to search for mates or patches of elevated food availability. Also, many of the plankton that generate a feeding current by vibrating appendages may in addition swim by using the same appendages in a different way (e.g., the nauplius larvae of most crustaceans) or by using other swimming appendages dedicated to propel themselves (most pelagic copepods and cladocerans).Whereas feeding and swimming may both compromise the survival of the organism, the trade-offs may be different. To get sufficient food, zooplankters need to daily clear a volume of water for prey that corresponds to about 10⁶ times their own body volume (20, 21) and hence, implicit in the feeding process is the need to examine or process large volumes of water. In contrast, dedicated swimming should translate the organism through the water as quietly as possible. Thus, we hypothesize that in microplankton, dedicated swimming produces flow fields that attenuate more readily and/or have a smaller spatial extension than the cases in which feeding and propulsion are intimately related.In this study we use particle image velocimetry (PIV) to describe the flow fields generated by micron- to millimeter-sized feeding and swimming zooplankton that use a variety of propulsion modes. We show that—across taxa and sizes—dedicated swimming produces flow fields with a much smaller spatial extension and a faster spatial attenuation than those produced by the plankton for which feeding and swimming are integrated, and we characterize the propulsion modes that minimize susceptibility to rheotactic predators.     

Catalpol protects against 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a well‐known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells. Catalpol inhibited TCDD‐induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3‐E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD‐treated cells and significantly inhibited TCDD‐induced increases in the levels of cytochrome P450 1A1 and extracellular signal‐regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal‐regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD‐induced damage in MC3T3‐E1 osteoblastic cells.     

Higher consumption of sugar-sweetened soft drinks increases the risk of hyperuricemia in Korean population: The Korean Multi-Rural Communities Cohort Study

Objective

The clinical implication of sugar-sweetened soft drinks on the risk of hyperuricemia has increased, especially in Western population studies. The aim of this study is to clarify the association between sugar-sweetened soft drinks and fruit drinks made from oranges and apples and the risk of hyperuricemia in the Korean Multi-Rural Communities Cohort.

Methods

A total of 9400 subjects were enrolled in the Korean Multi-Rural Communities Cohort Study, and a cross-sectional analysis was performed. Five quintiles (Q1–Q5) according to consumption of soft drinks and other fruit/fruit juices were classified and then categorized into three groups (Q1–Q3, Q4, and Q5) to assess the risk of hyperuricemia. Information on dietary intake was collected by well-trained interviewers using validated food frequency questionnaires.

Results

Higher consumption of sugar-sweetened soft drinks (Q5) increased the risk of hyperuricemia in males (adjusted OR = 1.35, 95% CI: 1.07–1.71) with a linear trend (p for trend = 0.01) and in females (adjusted OR = 1.40, 95% CI: 1.03–1.90) with no linear trend (p for trend = 0.09), compared to lower consumption (Q1–Q3). However, there were no significant differences of serum uric acid level according to the three categories of soft drink consumption, Q1–Q3, Q3, and Q5, in males (p = 0.21) or in females (p = 0.16), whereas all subjects showed statistical significance of serum uric acid level within the categories (p < 0.001). Estimated amount of soft drink intake was associated with serum uric acid level in males (β = 0.001; p = 0.01) but not in females (β = 0.0005; p = 0.10).

Conclusion

Higher consumption of sugar-sweetened soft drinks increased the risk of hyperuricemia in the Korean population, showing a differential linear trend for hyperuricemia according to gender.