KDM4 Inhibitors Research Articles

Targeting N-methyl-lysine histone demethylase KDM4 in cancer: natural products inhibitors as a driving force for epigenetic drug discovery.

KDM4A-F enzymes are a subfamily of histone demethylases containing the Jumonji C domain (JmjC) using Fe(II) and 2-oxoglutarate for their catalytic function. KDM4 enzymes are overexpressed or deregulated in cancer, thus leading to alteration in chromatin structure and transcriptional defects. As KDM4 enzymes have been associated with malignancy, they may represent novel targets for developing innovative therapeutic tools to treat different solid and blood tumors, of note, KDM4A is the isozyme most frequently associated with aggressive phenotypes of these tumors. To this aim, industrial and academic medicinal chemistry efforts have identified different KDM4 inhibitors. In most cases, these are pan-KDM4 inhibitors, with low selectivity against other Jumonji family members. The pharmacophoric features of the inhibitors frequently include a chelating group capable of coordinating the catalytic iron within the active site of the KDM4 enzyme. Nonetheless, non-chelating compounds have also demonstrated promising inhibitory activity, suggesting potential flexibility in the drug design. Several natural products, containing monovalent or bivalent chelators, have been identified as KDM4 inhibitors, albeit with a micromolar inhibition potency. This highlights the potential for leveraging them as templates for the design and synthesis of new derivatives, exploiting nature's chemical diversity to pursue more potent and selective KDM4 inhibitors.

Jumonji histone demethylases are therapeutic targets in small cell lung cancer

Small cell lung cancer (SCLC) is a recalcitrant cancer of neuroendocrine (NE) origin. Changes in therapeutic approaches against SCLC have been lacking over the decades. Here, we use preclinical models to identify a new therapeutic vulnerability in SCLC consisting of the targetable Jumonji lysine demethylase (KDM) family. We show that Jumonji demethylase inhibitors block malignant growth and that etoposide-resistant SCLC cell lines are particularly sensitive to Jumonji inhibition. Mechanistically, small molecule-mediated inhibition of Jumonji KDMs activates endoplasmic reticulum (ER) stress genes, upregulates ER stress signaling, and triggers apoptotic cell death. Furthermore, Jumonji inhibitors decrease protein levels of SCLC NE markers INSM1 and Secretogranin-3 and of driver transcription factors ASCL1 and NEUROD1. Genetic knockdown of KDM4A, a Jumonji demethylase highly expressed in SCLC and a known regulator of ER stress genes, induces ER stress response genes, decreases INSM1, Secretogranin-3, and NEUROD1 and inhibits proliferation of SCLC in vitro and in vivo. Lastly, we demonstrate that two different small molecule Jumonji KDM inhibitors (pan-inhibitor JIB-04 and KDM4 inhibitor SD70) block the growth of SCLC tumor xenografts in vivo. Our study highlights the translational potential of Jumonji KDM inhibitors against SCLC, a clinically feasible approach in light of recently opened clinical trials evaluating this drug class, and establishes KDM4A as a relevant target across SCLC subtypes.

Abstract A050: A Phase 1 dose escalation study of TACH101, a first-in-class KDM4 inhibitor for advanced solid tumors

Abstract The KDM4 family of histone demethylases (KDM4A, B, C, D) have been identified as key oncogenic drivers in various cancers. KDM4 proteins control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histones H3K9 and H3K36 and have been shown to play an important role in epigenetic dysregulation during tumorigenesis. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform is not effective. TACH101 is a novel, first-in-class small molecule KDM4 inhibitor that targets isoforms A-D. In vitro and in vivo studies have shown potent anti-proliferative activity and strong tumor growth inhibition across multiple xenograft models. Preclinical studies demonstrated favorable pharmacokinetics (PK) and safety profiles. Initial clinical development of TACH101 will target advanced or metastatic solid tumors. TACH101-CS-0001 is an open-label, single-arm, phase 1 dose escalation study evaluating the safety and tolerability of orally administered TACH101 in subjects with solid tumors. Patients receive TACH101 in a single-dose lead-in period followed by repeated dosing on a weekly schedule. Using a Bayesian optimal interval (BOIN) design, the trial will evaluate up to 8 dose levels following a 3-days on/4-days off dosing schedule. Escalation across 2 dose cohorts (5 and 10mg) has been completed and enrollment into cohort 3 (25 mg) has begun. The planned enrollment is up to 33 patients including dose escalation and dose expansion (highest safe dose level) cohorts. Inclusion criteria include advanced or metastatic solid tumor that has progressed or was non-responsive or intolerant to available therapies and for which no standard or available curative therapy exists. Patients must have disease that is measurable according to RECIST (v1.1) and an ECOG performance status score of 0 to 1. Exclusion criteria include severe hematologic, hepatic, or renal insufficiency. Primary endpoints will assess safety and tolerability to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). Secondary endpoints will evaluate PK and preliminary anti-tumor activity as assessed by objective response rate (ORR), duration of response (DOR), and clinical benefit rate (CBR). Exploratory endpoints include evaluation of circulating tumor DNA (ctDNA)-based genetic markers as well as molecular characterization of optional tissue biopsies. This trial will be the first to evaluate the full potential of KDM4 inhibition in solid tumors with the novel epigenetic-targeting drug candidate TACH101 in subjects with advanced or metastatic cancers. (NCT05076552). Citation Format: Apostolia Tsimberidou, Andrae L Vandross, David Sommerhalder, Charmaine Joseph, Chandtip Chandhasin, Frank Perabo, Michael F Clarke. A Phase 1 dose escalation study of TACH101, a first-in-class KDM4 inhibitor for advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A050.

KDM4 Demethylases: Structure, Function, and Inhibitors.

KDM4 histone demethylases mainly catalyze the removal of methyl marks from H3K9 and H3K36 to epigenetically regulate chromatin structure and gene expression. KDM4 expression is strictly regulated to ensure proper function in a myriad of biological processes, including transcription, cellular proliferation and differentiation, DNA damage repair, immune response, and stem cell self-renewal. Aberrant expression of KDM4 demethylase has been documented in many types of blood and solid tumors, and thus, KDM4s represent promising therapeutic targets. In this chapter, we summarize the current knowledge of the structures and regulatory mechanisms of KDM4 proteins and our understanding of their alterations in human pathological processes with a focus on development and cancer. We also review the reported KDM4 inhibitors and discuss their potential as therapeutic agents.

Recent Advances with KDM4 Inhibitors and Potential Applications.

The histone lysine demethylase 4 (KDM4) family plays an important role in regulating gene transcription, DNA repair, and metabolism. The dysregulation of KDM4 functions is associated with many human disorders, including cancer, obesity, and cardiovascular diseases. Selective and potent KDM4 inhibitors may help not only to understand the role of KDM4 in these disorders but also to provide potential therapeutic opportunities. Here, we provide an overview of the field and discuss current status, challenges, and opportunities lying ahead in the development of KDM4-based anticancer therapeutics.

KDM4C Contributes to Trophoblast-like Stem Cell Conversion from Porcine-Induced Pluripotent Stem Cells (piPSCs) via Regulating CDX2.

Studies on ESRRB-regulating porcine-induced pluripotent stem cells (piPSCs) converted to trophoblast-like stem cells (TLSCs) contribute to the understanding of early embryo development. However, the epigenetic modification regulation network during the conversion is poorly understood. Here, the global change in histone H3 Lysine 4, 9, 27, 36 methylation and Lysine 27 acetylation was investigated in piPSCs and TLSCs. We found a high modification profile of H3K36me2 in TLSCs compared to that of piPSCs, whereas the profiles of other modifications remained constant. KDM4C, a H3K36me3/2 demethylase, whose gene body region was combined with ESRRB, was upregulated in TLSCs. Moreover, KDM4 inhibitor supplementation rescued the AP-negative phenotype observed in TLSCs, confirming that KDM4C could regulate the pluripotency of TLSCs. Subsequently, KDM4C replenishment results show the significantly repressed proliferation and AP-positive staining of TLSCs. The expressions of CDX2 and KRT8 were also upregulated after KDM4C overexpression. In summary, these results show that KDM4C replaced the function of ESRRB. These findings reveal the unique and crucial role of KDM4C-mediated epigenetic chromatin modifications in determination of piPSCs’ fate and expand the understanding of the connection between piPSCs and TSCs.

Abstract 3720: TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the US accounting for about 22% of newly diagnosed cases of B-cell NHL. It is considered to be more aggressive and to have worse prognosis due to low response to existing treatments. KDM4 is a family of histone demethylases that can drive tumor growth by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 alters post-translational histone modification and is associated with many types of cancer, including DLBCL. TACH101 is a novel, potent small molecule inhibitor of KDM4 that is being developed for treatment of advanced cancers, including DLBCL. Methods: TACH101 was evaluated in vitro and in vivo using cancer cell lines and patient-derived organoid (PDO) and xenograft (PDX) models of DLBCL. Results: TACH101 is a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values < 0.100 μM for all four isoforms. An initial screen using a 301-cell line panel showed that DLBCL cell lines are sensitive to TACH101 (IC50 < 10 nM). In an additional panel of DLBCL cell lines, TACH101 inhibited proliferation in a dose-dependent manner in all DLBCL cell lines, independent of molecular subtype, with mean IC50’s of 0.03 ± 0.01 μM in ABC DLBCL cell lines (n=6); 0.02 ± 0.01 μM in GCB DLBCL cell lines (n=7) and 0.02 ± 0.01 μM in PMBL DLBCL cell lines (n=2). In OCI-LY19 DLBCL xenografts in vivo, TACH101 was well tolerated and inhibited tumor growth by 55% to 100%, depending on dosing regimen (either QD or BID following a 3 days on/4 days off schedule). In PK studies, TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzymes. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human studies. Conclusions: The KDM4 inhibitor, TACH101, had compelling activity in preclinical DLBCL models, suggesting that TACH101 could be an effective therapy for DLBCL. Preparations to advance the drug into clinical trials are underway. Citation Format: Frank Perabo, Chandtip Chandhasin, Sanghee Yoo, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3720.

TACH101, a first-in-class pan-inhibitor of KDM4 for treatment of gastrointestinal cancers.

132 Background: TACH101 is a novel, potent small molecule inhibitor of KDM4, a novel epigenetic target for cancer therapy. KDM4 is a family of histone lysine demethylases that, when overexpressed, drives key processes linked to cancer. Validation of KDM4 as a driver gene was confirmed across gastrointestinal tumor types including esophageal, colon and gastric cancers, and is associated with formation of aggressive tumors. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell inhibition assays, patient-derived xenograft (PDX) and organoid models, and bioinformatics analyses studies. Results: In vitro, TACH101 treatment potently inhibited cell proliferation in cell lines and organoid models representing esophageal, CRC, and gastric cancers. TACH101 induced apoptosis in human CRC (HT-29) and esophageal (KYSE-150) cancer cell lines (EC50s 0.033 - 0.092 µM). Further evaluation using a panel of > 300 cell lines from different tumor types showed potent activity of TACH101 against gastric cancer and CRC. In gastric cancer, 2D cell viability inhibition assays conducted on a panel of 11 gastric cancer cell lines showed 9/11 (82%) were sensitive to TACH101 treatment (IC50 0.004 - 0.072 µM); in PDX models, 4/5 (80%) were sensitive to TACH101 treatment (IC50 0.007 - 0.039 µM). In CRC, bioinformatics analysis indicated increased TACH101 sensitivity in cell lines with MSI-H status (IC50 1-150 nM). Sensitivity of MSI-H CRC to TACH101 was further confirmed in a panel of 14 CRC PDX models and 7 CRC organoid models in culture-based viability inhibition assays. In PDX models, 5/5 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.001 - 0.014 µM), whereas 4/8 (50%) characterized as MSS were sensitive to TACH101 (IC50 0.003 - 0.270 µM). In patient derived CRC organoid models, 3/3 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.022 - 0.149 µM) whereas 0/3 (0%) characterized as MSS were sensitive (IC50 > 10 µM). In vivo, TACH101 triggered effective tumor control (≥70%) in xenograft models of CRC (SU60), esophageal (KYSE-150) and gastric (GXA-3036) cancers. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Preclinical work on TACH101 KDM4 inhibitor demonstrates compelling data and applicability as a potential therapy for gastrointestinal cancers. Preparations to advance TACH101 into clinical trials are underway.

KDM4 inhibitor SD49-7 attenuates leukemia stem cell via KDM4A/MDM2/p21CIP1 axis.

Rationale: Traditional treatments for leukemia fail to address stem cell drug resistance characterized by epigenetic mediators such as histone lysine-specific demethylase 4 (KDM4). The KDM4 family, which acts as epigenetic regulators inducing histone demethylation during the development and progression of leukemia, lacks specific molecular inhibitors.Methods: The KDM4 inhibitor, SD49-7, was synthesized and purified based on acyl hydrazone Schiff base. The interaction between SD49-7 and KDM4s was monitored in vitro by surface plasma resonance (SPR). In vitro and in vivo biological function experiments were performed to analyze apoptosis, colony-formation, proliferation, differentiation, and cell cycle in cell sub-lines and mice. Molecular mechanisms were demonstrated by RNA-seq, ChIP-seq, RT-qPCR and Western blotting.Results: We found significantly high KDM4A expression levels in several human leukemia subtypes. The knockdown of KDM4s inhibited leukemogenesis in the MLL-AF9 leukemia mouse model but did not affect the survival of normal human hematopoietic cells. We identified SD49-7 as a selective KDM4 inhibitor that impaired the progression of leukemia stem cells (LSCs) in vitro. SD49-7 suppressed leukemia development in the mouse model and patient-derived xenograft model of leukemia. Depletion of KDM4s activated the apoptosis signaling pathway by suppressing MDM2 expression via modulating H3K9me3 levels on the MDM2 promoter region.Conclusion: Our study demonstrates a unique KDM4 inhibitor for LSCs to overcome the resistance to traditional treatment and offers KDM4 inhibition as a promising strategy for resistant leukemia therapy.

KDM4 Involvement in Breast Cancer and Possible Therapeutic Approaches.

Breast cancer (BC) is the second leading cause of cancer death in women, although recent scientific and technological achievements have led to significant improvements in progression-free disease and overall survival of patients. Genetic mutations and epigenetic modifications play a critical role in deregulating gene expression, leading to uncontrolled cell proliferation and cancer progression. Aberrant histone modifications are one of the most frequent epigenetic mechanisms occurring in cancer. In particular, methylation and demethylation of specific lysine residues alter gene accessibility via histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs). The KDM family includes more than 30 members, grouped into six subfamilies and two classes based on their sequency homology and catalytic mechanisms, respectively. Specifically, the KDM4 gene family comprises six members, KDM4A-F, which are associated with oncogene activation, tumor suppressor silencing, alteration of hormone receptor downstream signaling, and chromosomal instability. Blocking the activity of KDM4 enzymes renders them “druggable” targets with therapeutic effects. Several KDM4 inhibitors have already been identified as anticancer drugs in vitro in BC cells. However, no KDM4 inhibitors have as yet entered clinical trials due to a number of issues, including structural similarities between KDM4 members and conservation of the active domain, which makes the discovery of selective inhibitors challenging. Here, we summarize our current knowledge of the molecular functions of KDM4 members in BC, describe currently available KDM4 inhibitors, and discuss their potential use in BC therapy.

210P TACH101, a first-in-class KDM4 inhibitor for treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents nearly 20% of all breast cancers and is considered to be more aggressive and to have poorer prognosis due to lack of effective treatments. KDM4 is a family of histone demethylases shown to drive cancer by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 leading to mistakes in post-translational histone modification is associated with many. cancers, including TNBC.

Abstract 2128: TACH101, a first-in-class pan inhibitor of KDM4 histone lysine demethylases

Abstract Background: Alterations in epigenetic control can cause defective post-translational histone modification and dysregulation of gene activity leading to cancer development. Overexpression of KDM4, a demethylase of histone lysine 9 methyl 2/3 and lysine 36 methyl 2/3 (H3K9me2/3 and H3K36me2/3), is documented in a variety of cancers and is linked to aggressive disease and poor clinical outcomes. TACH101 is a novel first-in-class, selective and potent pan KDM4 inhibitor with favorable pharmacologic properties. Methods: TACH101 was evaluated using in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines and patient-derived organoid models, apoptotic and cell cycle analyses, pharmacology studies in various animal species, and efficacy studies in xenograft tumor models. Results: Inhibition mechanism studies demonstrated TACH101 to be a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values less than 0.100 μM for all four isoforms. In vitro, TACH101 demonstrated potent increase of H3K36me3 levels (EC50 <0.001 μM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and showed potent anti-proliferative activity in multiple cell lines in OncoPanel. Sub-micromolar levels of TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. TACH101 effects on cell cycle demonstrated a dose-dependent increase in the proportion of cells in S-phase at 24 and 48 hours relative to control: TACH101 at 0.01 μM concentration yielded fold increase of 1.7 and 2.1, and at 0.1 μM concentration yielded fold increase of 2.1 and 3.2. In vivo, TACH101 triggered effective tumor growth control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Flow cytometric analysis performed on single cells isolated from vehicle and TACH101-treated tumors showed a significant 2.5-fold reduction in population of tumorigenic cells (CD44high; EpCAM+) compared to vehicle control, indicating TACH101 may prevent self-renewal of cancer stem cells. This was corroborated in a study evaluating TACH101 effects on tumor initiating cell (TIC) frequency in colorectal tumors where TACH101 treatment reduced TIC frequency by 4.4-fold. Pharmacologic studies showed plasma protein binding of TACH101 to be ≥99% bound in mouse, rat, dog, and human. PK studies showed TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzyme activities. Conclusions: TACH101 is a potent pan inhibitor of KDM4 that is suggestive of broad potential in cancer treatment. Further preclinical evaluation is ongoing to advance the molecule into clinical trials. Citation Format: Sanghee Yoo, Chandtip Chandhasin, Joselyn R. Del Rosario, Young K. Chen, Jeff Stafford, Stephen Quake, Frank Perabo, Michael F. Clarke. TACH101, a first-in-class pan inhibitor of KDM4 histone lysine demethylases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2128.

Inhibition of histone lysine demethylases with TACH101, a first-in-class pan-inhibitor of KDM4.

3105 Background: The KDM4 family of histone lysine demethylases consists of four main isoforms (KDM4A, B, C, D), all of which have been identified as key oncogenic drivers. They function as epigenetic regulators and control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play an important role in the epigenetic dysregulation in various cancers and is linked to more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform appears to not be effective. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Here we present data that show TACH101 has promising pre-clinical and pharmacologic properties as a cancer therapeutic. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines, apoptotic and cell cycle analyses, and efficacy studies in various xenograft tumor models and patient-derived organoid models. Results: In vitro, TACH101 was broadly effective in killing 67% (200 out of 300) of cancer cell lines screened. TACH101 demonstrated potent increase of H3K36me3 levels (EC50 < 0.001 mM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and potent anti-proliferative activity in multiple cell lines in OncoPanel. TACH101 treatment increased cancer cell population in S-phase in multiple cancer cell lines indicating cell-cycle arrest. TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. In vivo, TACH101 triggered effective tumor control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Further evaluation using a panel of patient-derived colorectal models and patient-derived organoids showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). TACH101 also reduced tumorigenic potential by 4.4-fold as determined by FACS analysis using sorted CD44High EpCAM+ population in Limiting Dilution Assays in vivo, suggesting that reduction of cancer stem cells by TACH101 may be effective in therapy-resistant settings. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Extensive preclinical work on TACH101 KDM4 inhibitor shows compelling data and broad applicability as a potential anti-cancer agent. Further evaluation is ongoing to advance the molecule into clinical trials.

Pharmacologic characterization of TACH101, a first-in-class KDM4 inhibitor for development as a cancer therapeutic.

e15067 Background: The histone Lysine (K) Demethylase 4 family, KDM4 family, is involved in nuclear functions such as programming development, activation or repression of transcription, timing and control of the cell cycle, and initiating DNA replication and repair through chromatin remodeling. Overexpression of KDM4 leading to mistakes in post-translational histone modification has been associated with many cancer types and is being investigated as a potential therapeutic drug target. TACH101 is a novel potent and small molecule inhibitor of the KDM4 family. It demonstrated strong efficacy in multiple cancer types in vitro and in vivo and favorable pharmacokinetics (PK) and safety profiles in preclinical models. Methods: The target interaction properties of TACH101 were evaluated using purified enzymes for biochemical, biophysical, and inhibition mechanism studies. For PK parameter measurements, Sprague-Dawley rats and Beagle dogs were used for intravenous or oral administration of TACH101. Hepatocytes or liver microsomes were used to measure cytochrome P450 (CYP) inhibition and induction studies. Cell lines expressing cardiac channels were used for radioligand displacement assays and patch clamping. Results: Biochemical, biophysical, and inhibition mechanism studies using purified enzymes showed TACH101 is a reversible, alpha-KG (co-factor) competitive, and slow-binding inhibitor with IC50 values less than 100 nM. Pharmacologic studies showed plasma protein binding of TACH101 to be ≥99% bound in mouse, rat, dog, and human. TACH101 appeared to be selective in off-target panel assays of receptors, ion channels and transporters. TACH101 did not show any significant binding displacement activity against cardiac ion channels or any effect on hERG in CHO cells using manual patch clamp techniques. In rats and dogs, TACH101 exhibited low systemic clearance, a moderate volume of distribution in rats and dogs, linear exposure profiles with a terminal half-life (̃ 6-hour) and high bioavailability (46-100%). In repeated dose studies in rats and dogs, mean Cmax and AUC values increased in a dose-related manner over the dose range evaluated. There was no significant impact of food on systemic exposure in dogs. TACH101 did not show inhibition or induction of CYP enzymes tested. Conclusions: TACH101 is a potent KDM4 specific inhibitor without significant off-target activity in in vitro and in vivo studies. It did not show inhibition or induction of CYP enzymes suggesting low probability of CYP mediated drug-drug interaction. The exposure from oral administration in rats and dogs was dose proportional and was not significantly affected by food intake in dogs. Given the favorable target activity, PK properties and safety profile, a Phase 1 study is being planned to advance TACH101 for patients with advanced cancer.

Advances in histone demethylase KDM4 as cancer therapeutic targets.

The KDM4 subfamily H3K9 histone demethylases are epigenetic regulators that control chromatin structure and gene expression by demethylating histone H3K9, H3K36, and H1.4K26. The KDM4 subfamily mainly consists of four proteins (KDM4A-D), all harboring the Jumonji C domain (JmjC) but with differential substrate specificities. KDM4A-C proteins also possess the double PHD and Tudor domains, whereas KDM4D lacks these domains. KDM4 proteins are overexpressed or deregulated in multiple cancers, cardiovascular diseases, and mental retardation and are thus potential therapeutic targets. Despite extensive efforts, however, there are very few KDM4-selective inhibitors. Defining the exact physiological and oncogenic functions of KDM4 demethylase will provide the foundation for the discovery of novel potent inhibitors. In this review, we focus on recent studies highlighting the oncogenic functions of KDM4s and the interplay between KDM4-mediated epigenetic and metabolic pathways in cancer. We also review currently available KDM4 inhibitors and discuss their potential as therapeutic agents for cancer treatment.

Abstract B015: Targeting histone demethylase KDM4 for cancers driven by chimeric transcription factors

Abstract Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target for cancer therapy as they usually do not bear druggable pockets. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood soft tissue sarcoma, mainly driven by the pathognomonic PAX3-FOXO1 or its variant PAX7-FOXO1 fusion oncoprotein. Even with intensive chemotherapy and radiotherapy, the 5-year survival for fusion positive aRMS is poor with no significant improvement in over recent decades. Here we show that PAX3-FOXO1 function is dependent on histone demethylase KDM4B, a potential therapeutic vulnerability for aRMS. KDM4B is highly expressed in PAX3-FOXO1-positive aRMS and is associated with late stage disease. KDM4B physically interacts with PAX3-FOXO1 via its JmjN and TUDOR domains. Genetic depletion and pharmacologic inhibition of KDM4B with a novel inhibitor QC6352 significantly delay tumor growth, impact the expression of FAX3-FOXO1 targets such as MYOD1 and FGFR4, and reduce chromatin accessibility of PAX3-FOXO1 target genes in accordance with the changes of KDM4B substrates such as H3K9me3. Combination of QC6352 with chemotherapy lead to significant xenograft regression and extended mouse survival in cell line- and PDX- based preclinical aRMS models. In summary, we have identified a targetable epigenetic modifier required for the functions of PAX3-FOXO-driven aRMS, providing pre-clinical data for a clinal trial combining the KDM4 inhibitor with standard chemotherapy for high-risk aRMS. Citation Format: Jun Yang, Shivendra Singh, Ahmed Abu-Zaid, Jie Fang, Andrew Davidoff. Targeting histone demethylase KDM4 for cancers driven by chimeric transcription factors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B015. doi:10.1158/1535-7163.TARG-19-B015

Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Can be Therapeutically Targeted Inducing Apoptotic Cell Death in a Caspase-Dependent Way

Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Can be Therapeutically Targeted Inducing Apoptotic Cell Death in a Caspase-Dependent Way

Abstract 155: KDM4 inhibition targets breast cancer stem-like cells

Abstract Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSC regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Citation Format: Jochen Maurer. KDM4 inhibition targets breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 155.

KDM4 Inhibition Targets Breast Cancer Stem-like Cells.

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Cancer Res; 77(21); 5900-12. ©2017 AACR.

Design of KDM4 Inhibitors with Antiproliferative Effects in Cancer Models.

Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound 6 (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.