Oral cancers: Lysine-specific demethylase 1 might help limit a tumor’s development and spread

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The most common head and neck cancer – oral squamous cell carcinoma – often starts off, as many other cancers do, quite innocently. Perhaps as a little white patch in the mouth or a small red bump on the gums. Easy to ignore, to downplay. But then something changes, and the little blotch becomes more ominous, starts growing, burrowing into connective tissue.

Patients who are lucky enough to see a dentist before things take a nasty turn have a shot at being able to prevent the lesions from turning cancerous—or can at least make sure treatment starts when it’s most effective. But for those who aren’t that lucky, the outlook can be bleak: the five-year survival rate of oral squamous cell carcinoma (OSCC) is around 66 percent. More than 10,000 Americans die of oral cancer every year; smokers and drinkers are hardest hit.

Now, researchers at Boston University’s Henry M. Goldman School of Dental Medicine have found that dialing back—or even genetically deleting – a protein that seems to spur the cancer’s growth might help limit a tumor’s development and spread. They say their findings make the protein, an enzyme called lysine-specific demethylase 1, a potential “druggable target” – something that doctors could aim chemo and immuno-oncology therapies at to take down a tumor. The study was published in February in Molecular Cancer Research.

Given that at least one-third of Americans don’t visit a dentist regularly, according to the Centers for Disease Control and Prevention, the discovery could be a future lifesaver for those who miss out on preventative care.

“These findings have significant implications for new and potentially more effective therapies for oral cancer patients,” says Manish V. Bais, a lead author on the study and SDM assistant professor of translational dental medicine. “This study is an important step toward the development of novel groundbreaking therapies to treat oral cancer.”

Maria Kukuruzinska, SDM’s associate dean for research and a coauthor on the study, says it was rare in the past for dental schools to be diving into the science behind head and neck cancers, with most of the research happening in cancer centers. But that’s changing and “dental schools have an advantage over traditional cancer centers when it comes to investigating the science behind the development of OSCC,” she says, “because we can get access to premalignant lesions, where cancer centers basically just see patients who are presenting with fully developed disease.”

Helping the body fight back: Anti-tumor immunity

Once OSCC takes hold, says Bais, there’s little chance of eliminating it completely. Clinicians can try chemotherapy and radiotherapy, even cutting out a tumor. “But there is no cure—you can shrink the tumor, but not eliminate it,” Bais says.

In previous research, Bais had found that lysine-specific demethylase 1 (LSD1)—an enzyme that typically plays a crucial role in normal cell and embryo development—goes out of control, or is “inappropriately upregulated,” in a range of cancers, including in the head and neck, as well as those in the brain, esophagus, liver, and lung.

“The expression of this enzyme goes up with each tumor stage,” says Bais, who’s also a member of BU’s Center for Multiscale & Translational Mechanobiology. “The worse the tumor, the higher the expression of this protein.”

In his lab, Bais began testing what would happen to tumors in the tongue if LSD1 was blocked. To restrict the enzyme, the researchers either knocked it out – by manipulating genes so LSD1 is effectively switched off – or used a type of drug called a small molecule inhibitor, which enters a cell and impedes its normal function.

Already in clinical trials for treating other cancers, small molecule inhibitors haven’t previously been tested against oral cancer. Bais found that disrupting LSD1 curbed the tumor’s growth.

“The aggressiveness, or bad behavior, of the tumor went down,” he says. “We found that when we inhibit this protein, it promotes anti-tumor immunity – our body tries to fight by itself.”

But LSD1 isn’t the only troublemaker in the tumor: when it’s upregulated, it messes with a cell communication process – the Hippo signaling pathway-YAP – that normally helps control organ growth and tissue regeneration. Bais says YAP, LSD1, and a couple of other proteins then get stuck in a vicious cycle, each one pushing the other into increasingly aggressive and harmful moves. “We need to break this cycle,” says Bais.

To find a new way of doing that, the researchers coupled the effort to inhibit LSD1 by targeting YAP with a different inhibitor, a drug called verteporfin. Originally developed to help treat serious eye conditions like macular degeneration, verteporfin is being tested by other researchers as a potential cancer treatment, including in ovarian cancer. The combination proved effective, according to Bais. He also threw a third drug into the mix.

Bais says using the LSD1 inhibitor in combination with a common immunotherapy drug that helps white blood cells in the immune system kill cancer cells – an immune checkpoint inhibitor called anti-Programmed Death 1 ligand antibody – “showed a favorable response.”

“Our findings provide a basis for future clinical studies based on the inhibition of LSD1, either as monotherapy or in combination with other agents to treat oral cancer in humans,” he says. The work was recently boosted with a new $2.6 million National Institute of Dental and Craniofacial Research grant. “Although our studies are preclinical, restricted to mice and some human tissue, we want to expand to look at human clinical trial samples.”

Predict success in humans

According to Kukuruzinska, Bais’ focus on the biology of oral cancer may also help make the development of other future treatments more efficient.

“People get very excited when you have a drug that may show some positive preliminary results, but very frequently, these studies move forward to humans, cost billions of dollars, and then eventually fail,” says Kukuruzinska, who’s also director of SDM’s predoctoral research program and a professor of translational dental medicine. “If you really understand what pathways, what cell processes are impacted by these inhibitors, then it allows you to predict in advance whether something is going to be successful in human patients.”

At BU, the dental school has a teaching clinic on site and shares a campus with the BU School of Medicine and its primary teaching hospital, Boston Medical Center.

It’s also home to BU’s Head & Neck Cancer Program – which pairs basic science researchers with clinicians to look at the underlying mechanisms of oral cancers – and Center for Oral Diseases, a multidisciplinary clinical-research collaborative.

“So, we can think about disease interception,” says Kukuruzinska. “And perhaps think about preventing the tumor from happening.”

With access to a clinic – as well as head and neck surgeons from the neighboring hospital – researchers like Bais can test any new treatments and approaches on human tissue samples.

“It’s a holy grail,” Kukuruzinska says of the human samples. “We can interrogate them for responses to small molecule inhibitors, by capturing tumor slices and trying to treat them with different inhibitors to see the response.”

Eventually, it could also open the door to personalized, precision medicine, with researchers trialing different therapies on tissue from individual patients. “And then it will predict whether this person can be treated with this study,” says Kukuruzinska. “This is something we really want to develop.”

With students involved in many of the research projects – three were coauthors on Bais’ paper and another, Thabet Alhousami (SDM’22), was a lead author—it means future dentists produced at BU will head into the clinic with a sharper eye for potential malicious bumps and blotches.

“They will be able to say, ‘This is precancerous or cancerous’ – it will impact their diagnoses,” says Bais. “Then, in terms of therapy, because they’re now aware of what can work, what immunotherapy can work, they can make specific reference to where patients should go next. It can improve the quality of diagnosis and treatment in the long term.”


LSD1 in cancer
LSD1 is involved in various stages of cancer, including development, progression, metastasis, and recurrence after therapy. Although overexpression of LSD1 has been reported in various cancer types and correlated with poor overall survival in patients [20–24], LSD1 does not appear to be a potent oncogene. Instead, LSD1 supports cancer progression by regulating gene expression in cancer cells in favor of adaptation to the tumor microenvironment. Further, LSD1 is considered a drug target in cancer, and numerous LSD1 inhibitors have been developed, some of which are currently undergoing clinical trials for the treatment of hematological cancers as well as lung cancer and other solid tumors [25]. LSD1 inhibitors are not within the scope of this work and have been thoroughly discussed in other reviews [25, 26]. We focus on several aspects of cancer wherein LSD1 plays a disease-promoting role, such as hypoxia, the epithelial-to-mesenchymal transition (EMT), cancer stemness and differentiation, as well as antitumor immunity.

Regulation of hypoxia by LSD1
Eukaryotic cells including cancer cells have an elaborate system for adaptation to low oxygen levels (hypoxia) in their microenvironment [27]. Hypoxia induces the transcriptional activation of various genes via the stabilization of hypoxia-inducible factor (HIF)-1α [27, 28]. In the normal physiological oxygen state (normoxia), a series of enzymatic reactions initiated by oxygen-sensing prolyl-hydroxylases (PHDs) maintain HIF-1α at low levels. PHD enzymes hydroxylate HIF-1α at its proline residues, which serves as a signal for its ubiquitylation by the von Hippel-Lindau (VHL)-containing E3 ubiquitin ligase complex [29] and subsequent proteasomal degradation. Under hypoxic conditions, the lack of oxygen inhibits PHD function, leading to HIF-1α stabilization, nuclear localization, and transcriptional activation. In addition to the oxygen-dependent regulation of HIF-1α stability, oxygen-independent mechanisms, such as the CHIP-HSP70 or RACK1-HSP90 pathways, are known to degrade HIF-1α [30, 31]. Furthermore, various post-translational modifications, including acetylation, methylation, and SUMOylation, are critical for the regulation of HIF-1α stability and function [32, 33].

The role of LSD1 as a positive factor in the hypoxic regulation of HIF-1α stability and transcriptional activity via demethylation of HIF-1α and HIF-1α-interacting protein RACK1 has been well studied [14, 34–36]. Monomethylation of the HIF-1α Lys32 residue by SET7/9 mediates HIF-1α ubiquitylation and degradation (Fig. 1A) [14, 34]. As the E3 ubiquitin ligase that is targeting monomethylated HIF-1α is not yet known, its identification adds to our understanding of HIF-1α stability regulation. Further, this E3 ubiquitin ligase may be a promising drug target for HIF-1α suppression via PROteolysis Targeting Chimera (PROTAC) technology [37, 38], especially in VHL-defective cancers. Under hypoxic conditions, LSD1 maintains HIF-1α stability and hypoxia-responsive gene expression by counteracting SET7/9-mediated HIF-1α monomethylation [14].

A mouse model harboring a lysine-to-alanine substitution at HIF-1α Lys32, which prevents its monomethylation, exhibited much higher HIF-1α levels, upregulated hypoxia-inducible gene expression, as well as enhanced tumor growth and angiogenesis [14], suggesting that monomethylation at this lysine residue is a critical regulator of HIF-1α function. Lysine monomethylation of HIF-1α also occurs at position 391 and is regulated by the interplay between SET7/9 and LSD1 (Fig. 1A) [35]. This site is in close proximity to the oxygen-dependent degradation domain (ODDD) of HIF-1α, and methylation by SET7/9 enhances VHL-mediated HIF-1α ubiquitylation [35]. LSD1 increases HIF-1α stability by inhibiting the methylation.

In addition, LSD1 prevents PHD2-induced hydroxylation and enhances K532 deacetylation (Fig. 1B) [35]. LSD1 increases MTA1 (Metastasis Associated 1, a component of NuRD complex) level as well as HDAC activity in NuRD complex, resulting in the decrease of Arrest defect 1 (ARD1)-mediated K532 acetylation. These events stabilize HIF-1α. Another level of LSD1-mediated HIF-1α stability regulation is through an oxygen-independent mechanism via demethylation of RACK1 (Fig. 1B) [36]. RACK1 was identified as a HIF-1α-interacting protein and was shown to recruit an E3 ubiquitin ligase independently of oxygen status [30].

LSD1 demethylates the Lys271 residue of RACK1, inhibiting its interaction with HIF-1α and thus stabilizing the latter [36]. Low cofactor FAD levels attenuate LSD1 demethylase activity during prolonged hypoxia and suppress HIF-1α through RACK1-mediated degradation [36]. In summary, LSD1 increases HIF-1α stability under hypoxic conditions via three mechanisms. First, direct demethylation of HIF-1α stabilizes it against methylation-mediated degradation. Second, LSD1 indirectly inhibits HIF-1α hydroxylation-mediated degradation. Third, LSD1 demethylates RACK1 and inhibits the RACK1-mediated degradation of HIF-1α. Thus, LSD1 inhibition is a promising strategy for preventing cancer cell adaptation to the hypoxic microenvironment.

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Fig. 1
Regulation of the stability of HIF-1α by LSD1 in hypoxia. A Methylation of HIF-1α Lys32 or Lys391 residue by SET7/9 induces ubiquitination and degradation of HIF-1α. LSD1 removes the corresponding methyl groups and thereby stabilizes HIF-1α. B LSD1 prevents PHD2-induced hydroxylation and enhances K532 deacetylation of HIF-1α, thereby stabilizing HIF-1α. In addition, demethylation of RACK1 by LSD1 inhibits the interaction of RACK1 with HIF-1α, consequently stabilizing HIF-1α

While most studies on the function of LSD1 in hypoxia have focused on its regulation of HIF-1α stability, Sakamoto et al. suggested that histone demethylation by LSD1 is partially involved in cancer cell metabolic reprogramming [39]. They observed a shift in the metabolic balance from glycolytic to mitochondrial respiration upon LSD1 depletion in hepatocellular carcinoma cells. Reduced glucose uptake and glycolytic activity were correlated with the decrease of HIF-1α levels, while elevated mitochondrial respiration in parallel to the increase of methylated H3K4 in the promoter region of respiratory genes. In various independent studies, LSD1 was shown to play a role in the cellular response to hypoxia [14, 35, 36, 39, 40]. Since LSD1 levels and activity were associated with angiogenesis, tumor growth [35], as well as the metabolic shift toward glycolysis [39], LSD1 inhibitors are promising candidates for suppressing cancer progression in relation to the hypoxic response.

LSD1 in the epithelial-to-mesenchymal transition (EMT)

The EMT is an essential process allowing solid cancer cells to gain migratory potential and relocate from their original location [41, 42]. The process involves repression of epithelial marker genes, such as E-cadherin, and the activation of mesenchymal marker genes, including vimentin. Various transcription factors involved in the EMT have been characterized, including the SNAIL, TWIST, and ZEB families [41, 42]. Among them, SNAIL is known to recruit a number of epigenetic regulators in order to establish the heterochromatin state in the promoter region of epithelial marker genes [43]. Further, LSD1 plays a critical role in the initiation of this epigenetic repressive state.

LSD1 was initially proposed as a negative regulator of EMT and invasion in breast cancer cells, acting through the inhibition of TGF-β signaling gene expression via a complex formed with NuRD [8]. However, many other reports later suggested a role of LSD1 in promoting EMT across various types of cancer. The EMT-promoting role of LSD1 was first described when it was identified as a SNAIL-binding protein [43, 44]. SNAIL interacts with LSD1 through the N-terminal SNAG domain and recruits LSD1 to target gene promoters (e.g., SNAIL gene promoter) via binding to E-box consensus sequences, where LSD1 strips H3K4me2 histone marks [43, 44]. LSD1 simultaneously recruits the CoREST repressor complex to the promoter for transcriptional repression [43].

Further, LSD1 contributes to the enhanced stability of the SNAIL protein preventing its GSK3β-mediated degradation via the ubiquitin–proteasome system [45]. Without LSD1 binding, SNAIL failed to repress target gene expression [43], highlighting the critical role of the former in SNAIL-mediated epithelial gene repression. SNAIL family member SLUG (also called SNAIL2) also interacts with LSD1 via its SNAG domain [46]. LSD1 together with SNAIL/SLUG were involved in the repression of cancer-related genes other than epithelial-specific genes depending on the cancer type. Examples included BRCA1 (Breast cancer 1; the tumor suppressor) in triple-negative breast cancer cells [46] and NDRG1 (N-myc downstream-regulated gene 1; the metastasis suppressor) in neuroblastoma with the MYCN oncogene amplification [47]. The contribution of SNAIL-LSD1 to the development of acute myeloid leukemia (AML), a hematological malignancy, suggests that the complex is also involved in aspects of cancer progression other than the EMT [48].

Genome-wide analysis of epigenomic reprogramming during the EMT confirmed the pivotal role of LSD1. TGF-β-induced EMT in mouse hepatocytes was accompanied by a reduction in heterochromatin mark H3K9me2 levels in parallel to an elevation in the euchromatin marks H3K36me3 and H3K4me3 [49]. Importantly, the major contributor to this reprogramming was found to be LSD1. In addition, the increased motility and chemoresistance of TGFβ-treated cells were LSD1-dependent [49]. Thus, LSD1 acts a critical epigenetic EMT regulator via both the repression of epithelial gene expression and the activation of mesenchymal gene expression. Genome-wide analysis revealed a general shift of chromatin structure toward a more open state with increased euchromatin histone marks, which was not in agreement with the repression of epithelial genes during the EMT. One possible scenario is that the repression of epithelial-specific expression occurred because of SNAIL/LSD1-induced local repressive chromatin states rather than a broader genomic state of repression.

Post-translational modification of LSD1 contributes to its activation during the EMT. Phosphorylation at the Ser112 residue is critical for EMT activation [50, 51]. In nude mice with LSD1 mutant-expressing MDA-MB-231 tumors, ectopic overexpression of wild-type LSD1 or a phosphorylation-mimicking LSD1-S111D (originally reported as S112 referring to mouse LSD1, while S111 is the human LSD1 residue) enhanced metastasis, whereas overexpression of a phosphorylation-defective S112A mutant did not change the metastatic potential of cancer cells [50]. The kinase responsible for phosphorylation at the site was suggested to be chromatin-anchored PKC-θ [51], although LSD1-S112 was originally identified as a target for PKCα in relation to circadian regulation [52] and inflammation [15]. LSD1, together with PKC-θ, localized to mesenchymal gene promoters and enhanced gene expression, while LSD1 inhibition repressed mesenchymal gene induction [51]. In contrast to the phosphorylation-mediated activation of LSD1 in EMT, acetylation was shown to negatively regulate EMT-promoting function of LSD1 in epithelial cells, thereby attenuating the EMT [53]. Acetyltransferase MOF, which is highly expressed in epithelial cells, acetylates LSD1 at multiple lysine residues, interfering with its association with chromatin and thus compromising the EMT-promoting function of LSD1 [53].

Since LSD1 was identified as a critical player in the EMT, various approaches have been employed for blocking the EMT via inhibition of either LSD1 activity or the SNAIL interaction [54, 55]. Structurally, the N-terminal SNAG domain of SNAIL mimics histone H3, allowing for its interaction with LSD1 [56]. Thus, a SNAG mimicry peptide blocked the SNAIL-LSD1 interaction [55]. In addition, LSD1 demethylase inhibitor tranylcypromine (Parnate) also suppressed their interaction [54, 55]. Further, the treatment of various cancer cell lines with these two inhibitory molecules resulted in an increased expression of E-cadherin and the suppression of motility and invasiveness [54, 55]. LSD1 inhibitors Pargyline and GSK-LSD1 were applied to restrict the EMT in prostate cancer cells and oral squamous cell carcinoma, respectively. These inhibitors activated epithelial genes and repressed mesenchymal gene expression, in turn suppressing or delaying progression [19, 57]. In summary, a considerable amount of evidence has confirmed the LSD1-mediated promotion of the EMT in various types of cancer. Accordingly, LSD1 inhibitors have been successfully applied to suppress the EMT process and cancer progression in these preclinical studies.

Maintenance of cancer stemness and regulation of differentiation by LSD1

The critical role of LSD1 in the regulation of stemness and differentiation was first reported in embryonic stem cells (ESCs) of humans and mice [58, 59]. LSD1 repressed the expression of several critical developmental genes by regulating the methylation status of H3K4 via its enzymatic activity [58]. As a result, LSD1 knockdown induced the differentiation of human ESCs via the early expression of mesodermal and endodermal marker genes, in parallel to increased levels of H3K4 di- and tri-methylation [58]. LSD1 depletion in mouse ESCs leads to their incomplete differentiation due to the partial silencing of ESC genes, as LSD1 was crucial for the decommissioning of ESC-specific enhancers during differentiation [59]. Many LSD1-regulated genes are its indirect targets, wherein LSD1 recruitment is mediated by master ESC transcription factors, including OCT4, SOX2, and NANOG [58, 59]. In addition to its role in ESCs, LSD1 is involved in the differentiation of adult stem cells in various tissues, including myogenic differentiation [60], adipogenesis [61], hematopoiesis [62, 63], and epithelial differentiation [64]. Taken together, LSD1 is a critical epigenetic factor that maintains the self-renewal potential of stem cells and regulates their cellular differentiation.

In tumors, a small population of cells containing stem cell-like properties (i.e., self-renewal, long-term growth, and drug resistance) have been identified and termed cancer stem cells (CSCs) [65, 66]. After the characterization of LSD1 as a pivotal regulator contributing to embryonic stem cell stemness and differentiation, various groups have explored its role in CSCs. LSD1 inhibition via small-molecule compounds selectively suppressed the growth of stem-like cancer cells in teratocarcinoma, embryonic carcinoma, and testicular seminoma, without significant growth inhibition observed in non-pluripotent cancer cells [67].

Since this initial observation, various studies have elucidated the function of LSD1 and the consequences of LSD1 deletion or pharmacological inhibition in CSCs from a variety of cancer types. Although the mechanism of LSD1 function in CSCs varies and has not been clearly established, it is related to the regulation of stemness and differentiation, similarly to its role in ESCs. Thus, LSD1 inhibition is to be accompanied by reduced stemness, cellular differentiation, and/or diminished drug resistance in CSCs.

Pharmacological LSD1 inhibition in small cell lung carcinoma (SCLC), a notoriously drug-resistant lung cancer type, had cytostatic effects with a delayed onset of growth both in vitro and in xenograft models [68]. LSD1 caused a change in the cellular state (i.e., inducing differentiation) via neuroendocrine marker gene expression changes, which are a molecular feature of SCLCs [68]. Unfortunately, not all SCLC cell lines respond to LSD1 inhibition, and the exact mechanism through which LSD1 contributes to the CSC phenotype has not been elucidated. In squamous cell carcinoma (SCC), LSD1 function was associated with the stem cell factor SOX2. LSD1 inhibitors selectively suppressed the growth and promoted the differentiation of SOX2-positive, but not SOX2-negative, SCCs, in conjunction with the upregulation of differentiation-associated genes [69].

In leukemia, LSD1 inhibition promotes cell differentiation. In MLL-AF9-driven leukemia, LSD1 sustained the expression of oncogenic genes maintaining stem cell potential in concert with the MLL-AF9 oncoprotein. Further, knockdown or pharmacological inhibition of LSD1 attenuated leukemia stem cell potential and induced differentiation [70]. Moreover, inhibition successfully induced cell fate transition in other types of leukemia. In particular, inhibiting LSD1 triggered the differentiation of non-acute promyelocytic leukemia (APL)-type AML cells, which do not respond to all-trans-retinoic acid (ATRA)-mediated differentiation, into ATRA-sensitive cells [71].

Interestingly, recent evidence revealed that even though LSD1 inhibition was not effective in APL cell treatment, it sensitized APL cells to physiological doses of retinoic acid so that combination treatment of LSD1 inhibitor and retinoic acid extended the survival of leukemic mice [72]. However, in this case, the demethylase activity of LSD1 was not correlated with its retinoic acid-mediated sensitizing ability, and LSD1 inhibition disrupted the interaction between LSD1 and GFI1 [72]. Pharmacological dissociation of LSD1 from the LSD1-GFI1 complex has been reported as crucial for the differentiation of AML cells [73, 74].

In addition, pharmacological inhibition of LSD1 in Merkel cell carcinoma, a primary neuroendocrine carcinoma of the skin, induced cell cycle arrest and cell fate change to the normal Merkel cell phenotype accompanied by the de-repression and activation of the neuronal transcription program [75]. Thus, LSD1 is a critical player in the maintenance of stemness, and LSD1 inhibition has been demonstrated as efficient for the treatment of differentiation-prone cancer types in pre-clinical models.

LSD1 is likely to regulate the methylation status of lineage- or cancer type-specific gene sets rather than the global chromatin methylation status in CSCs [64, 70, 71, 76]. The question is how LSD1 selectively targets gene-specific promoters. One plausible explanation is that LSD1 works together with or regulates stem cell factors in a subset of CSC types. One candidate factor, SOX2, was reported to cooperate with LSD1 in lung cancer [69], HER2-positive breast cancer [77], human ovarian teratocarcinoma [17], and pluripotent cancer cells, including teratocarcinoma, embryonic carcinoma, and seminoma CSCs [67].

LSD1 inhibition repressed the expression of SOX2 in lung squamous cell carcinomas, resulting in the suppression of SOX2-mediated oncogenic potential [69]. In human ovarian teratocarcinomas, SOX2 was susceptible to monomethylation-mediated degradation, which was reversed by LSD1, and thus LSD1 inhibition caused SOX2 destabilization [17]. LSD1 inhibition selectively blocked CSC-driven mammosphere formation in SOX2-driven CSCs [77].

Another stem cell factor, OCT4, was also suggested as a stemness-regulating factor working in concert with LSD1. The LSD1-OCT4 interaction in CSCs was suggested to maintain enhancers susceptible to reactivation, leading to the abnormal expression of pluripotency-related genes [78]. In LGR5-positive hepatocellular carcinoma, LGR5 and LSD1 appeared to enhance each other’s expression, maintaining the stem cell characteristics of carcinoma cells [79].

A key feature of neuroblastoma is the impaired neuronal differentiation in parallel to the high expression of myelin transcription factor 1 (MYT1). Knockdown of MYT1 induced neuro-differentiation in neuroblastoma cells [80]. Pharmacological inhibition of MYT1 interaction partner LSD1 had similar effects. Therefore, the roles of LSD1 in CSCs need to be considered together with those of its interaction partners, which are most likely lineage- or cancer type-specific transcriptional regulators.

LSD1 in anti-tumor immunity

The immune surveillance system of the human body continuously identifies and clears tumor cells through a multi-step process. In brief, these steps include the recognition of tumor-associated antigens by dendritic cells (DCs), antigen presentation via major histocompatibility complexes on the DC surface, the migration of DCs to lymphoid organs, DC-T cell contact resulting in T cell activation, relocation of activated T cells to peripheral tumor sites, and finally, the recognition and cytotoxic T cell-mediated destruction of cancer cells [81]. In addition, tumor antigen-specific B cells proliferate, differentiate, and produce antibodies to help with the destruction of cancer cells [81]. However, cancer cells employ various mechanisms to perturb and escape from immune surveillance, including immune editing [82]. Therefore, various approaches for overcoming the immune escape of cancer cells are being actively investigated.

Tumor immunotherapy is a major antitumor treatment approach that has recently emerged into the clinical spotlight. It was not long ago that LSD1 inhibitors were also shown to be effective in antitumor immunotherapy (Fig. 2). Based on the fact that targeting epigenetic regulators can boost the antitumor immune response, researchers screened compounds targeting chromatin factors that upregulate the expression of endogenous retroviral element (ERV)—as well as type I interferon (IFN)-responsive genes, and identified GSK-LSD1 as a hit [16]. Further analysis revealed that LSD1 inhibition caused ERV expression in parallel to H3K4me2 upregulation at various ERV regions, resulting in the downregulation of RNA-induced silencing complex (RISC), which otherwise clears double-stranded ERV transcripts.

These two phenomena in turn enhanced the cellular response to dsRNA by activating type I IFN signaling. In addition, LSD1 depletion or inhibition enhanced tumor immunogenicity, resulting in increased T cell infiltration of tumors. Furthermore, LSD1 inhibition sensitized PD-(L)1-resistant tumor cells to checkpoint blockade [16].

This was the first report to highlight the potential application of LSD1 inhibition in cancer immunotherapy, especially for the treatment of tumors that are poorly immunogenic and resistant to PD-(L)1 blockade. In agreement with this observation, various researchers reported the pro-immunogenic effects of LSD1 inhibition in tumors, which lead enhanced immune checkpoint blockade efficacy.

LSD1 inhibition in triple-negative breast cancer (TNBC) increased the expression of CD8+ T cell-attracting chemokines and PD-L1 in parallel to upregulated H3K4me2 marks at the respective gene promoter regions, resulting in enhanced CD8+ T cell infiltration at tumor sites [83]. Further, combination treatment with LSD1 inhibitor and an anti-PD-1 antibody in TNBC xenograft mice amplified PD-1 blockade efficacy [83]. Enhanced antitumor immune responses, including T cell infiltration, after LSD1 inhibition have also been reported in SWI/SNF-mutated ovarian cancers as well as in a 4T1 melanoma mouse model [84, 85].

Interestingly, LSD1 deficiency of CD8+ T cells in a murine melanoma model resulted in lower tumor growth, higher PD-1 levels in the tumor-infiltrating population, and no significant difference in overall health compared to wild-type mice [86]. CoREST complex inhibitor corin, which is derived from a class I HDAC inhibitor and an LSD1 inhibitor [87], induced the expression of proinflammatory cytokine genes in Tregs, resulting in increased CD8+ T cell tumor infiltration and reduced tumor burden [88]. Therefore, LSD1 inhibition seems to exert anti-tumor effects on both tumor and immune cells. Further, it is of interest to observe whether a single LSD1 inhibitor can exert similar effects on Treg cells as the dual CoREST inhibitor corin.

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Fig. 2
The effect of LSD1 inhibition on anti-tumor immunity. LSD1 inhibition in cancer cells enhances tumor immunogenicity and secretion of chemokines that attract T cells, while reversing the resistance to PD-(L)1 blockade. LSD1 inhibition upregulates pro-inflammatory cytokines in Treg cells, promotes the polarization of macrophages into the anti-tumor M1-like macrophages, and enhances the infiltration of these macrophages and CD8 + T cells into the tumor

In addition to modulating tumor immunogenicity and PD-(L)1 blockade, LSD1 inhibition potentiates antitumor immunity by activating innate immune cells, including macrophages and natural killer cells. In a xenograft model of MDA-MB-231 breast cancer in BALB/c nude mice, LSD1 inhibition via phenelzine resulted in increased infiltration of anti-tumor M1 macrophages in the tumor microenvironment [51]. Further analysis of the effect of LSD1 inhibitors on macrophage polarization programs revealed that phenelzine could target both FAD and CoREST binding domain, while catalytic inhibitor GSK2879552 could not switch the macrophage polarization program toward an anti-tumoral M1-like phenotype [89]. In contrast, catalytic inhibitor GSK-LSD1, but not scaffold inhibitors, increased natural killer cell-mediated tumor regression in a mouse model of pediatric high-grade glioma [90]. One concern was the cytostatic and cytotoxic effects of scaffold-type LSD1 inhibitors on natural killer cells, which did not affect T cells [90]. This was due to the impaired metabolism and glutathione depletion in inhibitor-treated natural killer cells, as glutathione supplementation rescued their cytolytic function [91].

Taken together, studies have thoroughly demonstrated the beneficial effects of LSD1 inhibition on antitumor immunotherapy. In cancer, LSD1 inhibition enhances tumor immunogenicity and the secretion of chemokines, while reversing resistance to PD-(L)1 blockade. Further, LSD1 inhibition increases proinflammatory cytokine expression in Treg cells and enhances tumor CD8+ T cell infiltration. Antitumor M1-like macrophage polarization and infiltration were also upregulated by LSD1 inhibition. As LSD1 inhibitors have therapeutic potential within immunotherapy, further in-depth research is crucial for determining which type of LSD1 inhibitors (catalytic inhibitor vs. scaffolding inhibitor) is to be used and what type of immunotherapy it should be combined with.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175190/


More information: Thabet Alhousami et al, Inhibition of LSD1 attenuates oral cancer development and promotes therapeutic efficacy of immune checkpoint blockade and Yap/Taz inhibition, Molecular Cancer Research (2022). DOI: 10.1158/1541-7786.MCR-21-0310

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