Entinostat

Scientists discovered a new target for reversible, non-hormonal male birth control. The drug, an HDAC inhibitor, blocked sperm production and fertility in male mice without affecting libido or future reproduction. Surveys show most men in the United States are interested in using male contraceptives, yet their options remain limited to unreliable condoms or invasive vasectomies. Recent attempts to develop drugs that block sperm production, maturation, or fertilization have had limited success, providing incomplete protection or severe side effects. New approaches to male contraception are needed, but because sperm development is so complex, researchers have struggled to identify parts of the process that can be safely and effectively tinkered with. Now, scientists at the Salk Institute have found a new method of interrupting sperm production, which is both non-hormonal and reversible. The study, published in Proceedings of the National Academy of Sciences (PNAS) on February 20, 2024, implicates a new protein complex in regulating gene expression during sperm production. The researchers demonstrate that treating male mice with an existing class of drugs, called HDAC (histone deacetylase) inhibitors, can interrupt the function of this protein complex and block fertility without affecting libido. "Most experimental male birth control drugs use a hammer approach to blocking sperm production, but ours is much more subtle," says senior author Ronald Evans, professor, director of the Gene Expression Laboratory, and March of Dimes Chair in Molecular and Developmental Biology at Salk. "This makes it a promising therapeutic approach, which we hope to see in development for human clinical trials soon." The human body produces several million new sperm per day. To do this, sperm stem cells in the testes continuously make more of themselves, until a signal tells them it's time to turn into sperm -- a process called spermatogenesis. This signal comes in the form of retinoic acid, a product of vitamin A. Pulses of retinoic acid bind to retinoic acid receptors in the cells, and when the system is aligned just right, this initiates a complex genetic program that turns the stem cells into mature sperm. Salk scientists found that for this to work, retinoic acid receptors must bind with a protein called SMRT (silencing mediator of retinoid and thyroid hormone receptors). SMRT then recruits HDACs, and this complex of proteins goes on to synchronize the expression of genes that produce sperm. Previous groups have tried to stop sperm production by directly blocking retinoic acid or its receptor. But retinoic acid is important to multiple organ systems, so interrupting it throughout the body can lead to various side effects -- a reason many previous studies and trials have failed to produce a viable drug. Evans and his colleagues instead asked whether they could modulate one of the molecules downstream of retinoic acid to produce a more targeted effect. The researchers first looked at a line of genetically engineered mice that had previously been developed in the lab, in which the SMRT protein was mutated and could no longer bind to retinoic acid receptors. Without this SMRT-retinoic acid receptor interaction, the mice were not able to produce mature sperm. However, they displayed normal testosterone levels and mounting behavior, indicating that their desire to mate was not affected. To see whether they could replicate these genetic results with pharmacological intervention, the researchers treated normal mice with MS-275, an oral HDAC inhibitor with FDA breakthrough status. By blocking the activity of the SMRT-retinoic acid receptor-HDAC complex, the drug successfully stopped sperm production without producing obvious side effects. Another remarkable thing also happened once the treatment was stopped: Within 60 days of going off the pill, the animals' fertility was completely restored, and all subsequent offspring were developmentally healthy. The authors say their strategy of inhibiting molecules downstream of retinoic acid is key to achieving this reversibility. Think of retinoic acid and the sperm-producing genes as two dancers in a waltz. Their rhythm and steps need to be coordinated with each other for the dance to work. But if you throw something in that makes the genes miss a step, the two are suddenly out of sync and the dance falls apart. In this case, the HDAC inhibitor causes the genes' misstep, halting the dance of sperm production. However, if the dancer can find its footing and get back in step with its partner, the waltz can resume. In the same way, the authors say that removing the HDAC inhibitor allows the sperm-producing genes to get back in sync with the pulses of retinoic acid, turning sperm production back on as desired. "It's all about timing," says co-author Michael Downes, a senior staff scientist in Evans' lab. "When we add the drug, the stem cells fall out of sync with the pulses of retinoic acid, and sperm production is halted, but as soon as we take the drug away, the stem cells can reestablish their coordination with retinoic acid and sperm production will start up again." The authors say the drug doesn't damage the sperm stem cells or their genomic integrity. While the drug was present, the sperm stem cells simply continued regenerating as stem cells, and when the drug was later removed, the cells could regain their ability to differentiate into mature sperm. "We weren't necessarily looking to develop male contraceptives when we discovered SMRT and generated this mouse line, but when we saw that their fertility was interrupted, we were able to follow the science and discover a potential therapeutic," says first author Suk-Hyun Hong, a staff researcher in Evans' lab. "It's a great example of how Salk's foundational biological research can lead to major translational impact." Other authors include Glenda Castro, Dan Wang, Russell Nofsinger, Annette R. Atkins, and Ruth T. Yu of Salk, Maureen Kane, Alexandra Folias, and Joseph L. Napoli of UC Berkeley, Paolo Sassone-Corsiof UC Irvine, Dirk G. de Rooij of Utrecht University, and Christopher Liddle of the University of Sydney. The work was supported by the National Institutes of Health (grants CA265762 and CA220468) and the Next Generation Sequencing and Flow Cytometry Cores at Salk, funded by the Salk Cancer Center (NCI grant NIH-NCI CCSG: P30 014195).

Researchers discovered how anti-cancer drugs called HDAC inhibitors help treat pancreatic cancer by altering scar tissue (fibroblast) development. The findings suggest HDAC inhibitors could be powerful tools in fighting pancreatic cancer -- alone and in combination with other therapies. Pancreatic cancer is one of the deadliest cancers -- only about one in eight patients survives five years after diagnosis. Those dismal statistics are in part due to the thick, nearly impenetrable wall of fibrosis, or scar tissue, that surrounds most pancreatic tumors and makes it hard for drugs to access and destroy the cancer cells. Now, researchers at the Salk Institute have discovered how a class of anti-cancer drugs called HDAC inhibitors can help treat pancreatic cancer by modulating the activation of fibroblasts -- the cells that make up that wall of scar tissue. The new research was published in Nature Communications on December 6, 2023. "These drugs turn out to be hitting both the tumor itself as well as the fibrotic tissue around it. This could be a very effective way to treat pancreatic cancers, which have typically been very difficult to reach," says senior author Professor Ronald Evans, director of Salk's Gene Expression Laboratory and March of Dimes Chair in Molecular and Developmental Biology. In response to a new pancreatic tumor, the pancreas typically activates fibroblasts -- the connective cells that support the structure of most organs. When flipped from a resting state to an active state, fibroblasts build a thick layer of scar tissue around the cancer. While this normal protective mechanism can help wall off a cancer and prevent its spread, fibroblasts also produce signaling molecules that the tumor itself takes advantage of to grow. "In the context of most pancreatic cancers, fibroblasts are acting as both good players and bad players," says Michael Downes, senior staff scientist and co-corresponding author on the paper. "It's a double-edged sword." In the new research, the team probed the effect on fibroblasts of an experimental class of cancer drugs known as histone deacetylase (HDAC) inhibitors. HDACs alter the three-dimensional structure of DNA inside cells, making some stretches of DNA easier or harder for other molecules to access and read. Targeting HDACs can therefore prevent cells from making large changes to their behavior, such as the out-of-control growth of cancer cells. But how the drugs work on all cell types is not well understood. In experiments on isolated cells, the researchers discovered that HDAC inhibitors prevented fibroblasts from becoming activated and tumor supportive. "Using HDAC inhibitors actually did two things -- it both turned down the growth signals from the fibroblasts to the cancer cells and it reduced the actual activation and accumulation of the fibroblasts," says Gaoyang Liang, first author and research associate in Evans' lab. In mice, the researchers found that one experimental HDAC inhibitor, entinostat, both reduced the activation of fibroblasts around pancreatic tumors and slowed tumor growth. When the researchers analyzed data from humans with pancreatic cancer, they discovered something similar: the higher the levels of HDAC1 in the fibrotic tissue around a patient's tumor, the worse their outcome. "This is in agreement with what we saw in cells and in mice," says Downes. "If you have more HDAC activities in the fibroblasts, you have a worse outcome. On the other hand, if you inhibit the HDACs, you have a better outcome." Since HDAC inhibitors work by preventing cells from activating certain genetic programs, the researchers wanted to know which stretches of DNA impacted by the drugs were most relevant for fibroblast activation. They identified several genes that HDAC inhibitors prevent from being expressed -- suggesting that new drugs could target those genes to keep fibroblasts from becoming activated and promoting cancer growth and fibrosis. "There have been some questions in the past about whether targeting fibroblasts is a good thing or a bad thing in pancreatic cancers, because people have shown that if you get rid of fibroblasts altogether it actually makes the cancers more aggressive," says Annette Atkins, co-author of the study and senior research scientist in Evans' lab. "But what our results suggest is that we don't have to get rid of them; just limiting their activation is beneficial." More work is needed to pinpoint how to best deliver HDAC inhibitors to the dense fibrotic tissue around pancreatic tumors, as well as how they might be most effectively combined with other cancer treatments. Other authors of the paper are Tae Gyu Oh, Nasun Hah, Yu Shi, Morgan L. Truitt, Corina E. Antal, Annette R. Atkins, Yuwenbin Li, Antonio F. M. Pinto, Dylan C. Nelson, Gabriela Estepa, Senada Bashi, Ester Banayo, Yang Dai, Ruth T. Yu, Tony Hunter, and Dannielle D. Engle of Salk; Hervé Tiriac of UC San Diego; Cory Fraser of HonorHealth Scottsdale; Serina Ng, Haiyong Han, and Daniel D. Von Hoff of The Translational Genomic Research Institute; and Christopher Liddle of the University of Sydney. The work was supported by grants from the Lustgarten Foundation (including award 552873), Don and Lorraine Freeberg Foundation, Ipsen Bioscience, a Stand Up To Cancer-Cancer Research UK-Lustgarten Foundation Pancreatic Cancer Dream Team Research Grant (SU2C-AACR-DT-20-16), a Ruth L. Kirschstein National Research Service Award (F32CA217033), a Life Sciences Research Foundation Fellowship, the Damon Runyon Cancer Research Foundation (DRG-2244-16), the National Institutes of Health (CA082683, 5T32CA009370), and the William Isacoff Research Foundation.