pinometostat (EPZ-5676)

Indeed, with molecular docking and biochemical experiments, we identified EPZ-5676 as a YTHDC1 inhibitor, and combination of EPZ-5676 with Cytarabine (Ara-c) significantly improved the efficacy of chemotherapy in B-ALL mouse models using YTHDC1high primary and lined B-ALL cells. Collectively, YTHDC1 is required for DDR in B-ALL cells by upregulating DDR-related gene expression via stabilizing m6A-modified KMT2C mRNA, thereby leading to increased histone H3K4 methylation, and targeted inhibition of YTHDC1 is a potentially new therapeutic strategy against B-ALL, especially YTHDC1high B-ALL.

Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

P1/2, N=6, Terminated, National Cancer Institute (NCI) | Phase classification: P1b/2 --> P1/2

Finally, drugs with high sensitivity to HNSCC (such as 5-Fluorouracil, Temozolomide, Carmustine, and EPZ5676) were explored and analyze the malignant characteristics of HNSCC. Malignant features associated with HNSCC include angiogenesis, EMT, and the cell cycle. This study has opened up new prospects for the prognosis, prediction, and clinical treatment strategy of HNSCC.

Notably, combination therapy with a mTORC1 inhibitor (Rapamycin) and a MA9/DOTL1 inhibitor (EPZ-5676) reduced the leukemia burden in MLL-AF9 mice lacking Celf2 in vivo. Our study elucidated a novel mechanism by which the CELF2/FAT10-AKT/mTORC1 axis regulates the proliferation of normal blood cells and the development of AML, thus providing potential therapeutic targets for myeloid leukemia suppression.

We performed cell viability experiments on leukemia cell lines (MV4; 11, MOLM13, RS4; 11, THP1, SEM, KOPN8, NOMO1, HL60, ML2) with single, two and three drug combinations using a menin inhibitor (VTP50469), a DOT1L inhibitor (EPZ5676), and a CDK9 inhibitor (AZD4573). Based on our preclinical in vitro findings, the co-inhibition of menin-DOT1L or menin-CDK9 is a promising approach for the treatment of MLL-r leukemia. The varying responses of different MLL-r leukemia cell lines to drug combination treatment indicate that not all cases of MLL-r leukemia will respond uniformly to treatment combinations. Taken together, our data provide a strategy to determine optimal treatment combinations for personalized treatment of MLL-r leukemia.

Concomitantly, these pinometostat-resistant cells showed upregulation of several myeloid-associated genes, including CD33 and LILRB4/CD85k. Taken together, this model comprehensively shows the adaptive potential of KMT2A-rearranged ALL cells upon losing dependency on one of its main oncogenic properties.

P1b/2, N=5, Terminated, National Cancer Institute (NCI) | N=37 --> 5 | Trial completion date: Jun 2023 --> Nov 2022 | Active, not recruiting --> Terminated | Trial primary completion date: Jun 2023 --> Nov 2022; Other - Study agent no longer available

As anticipated, pinometostat-resistant cells displayed a slight cross-resistance to most chemotherapeutic agents currently used in ALL treatments but became remarkably more sensitive toward the BCL-2 inhibitor venetoclax. Also, our model demonstrates that under prolonged pressure of DOT1L inhibition, KMT2A-rearranged ALL cells seem to initiate a reprogramming process that involves the acquirement (or selection) of myeloid-like characteristics, an ability that may be connected to leukemic lineage switches which are not uncommon in KMT2A-rearranged acute leukemias. Hence, our model represents an important tool to study the complex biology of KMT2A-rearranged leukemia, and its existence and availability requires to be shared with the community.

Inhibition of DOT1L's methyltransferase activity by the small molecule inhibitor (DOT1Li) EPZ-5676 also effectively targeted ovarian CSCs...Targeting DOT1L in OC could be a new strategy to eliminate CSCs. Implications: This study found that the histone methyltransferase DOT1L regulates the self-renewal and tumor initiation capacity of ovarian CSCs and suggests DOT1L as a new cancer target.

The most potent compound 12 exhibited comparable anticancer cellular activities to those of EPZ5676, a clinical stage enzymatic inhibitor of DOT1L in several leukemia cell lines containing MLL fusion proteins. Mechanism studies for compound 12 indicated that it did not affect the global methylation of H3K79 catalyzed by DOT1L but could effectively suppress the methylation of H3K79 at MLL fusion proteins targeted genes and inhibit the expressions of these genes. Our studies thus demonstrated that inhibiting the protein-protein interactions between DOT1L and MLL fusion proteins is a potentially effective strategy for the treatment of MLL rearranged leukemias.

P1b/2, N=37, Active, not recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2022 --> Jun 2023 | Trial primary completion date: Jun 2022 --> Jun 2023

DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell enriched TNBC.

Moreover, one DOT1L inhibitor, EPZ-5676, has entered clinical trials, but its clinical activity is modest...However, drug R&D strategies and platforms need to be developed and preclinical experiments need to be performed with the purpose of blocking DOT1L-associated PPIs. DOT1L epigenetic-based combination therapy is worth considering and exploring, but the therapy should be based on a thorough understanding of the regulatory mechanism of DOT1L epigenetic modifications.

Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

P1b/2, N=37, Active, not recruiting, National Cancer Institute (NCI) | Recruiting --> Active, not recruiting

Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia.

Pharmacological inhibition of DOT1L (EPZ-5676, EPZ004777, and SGC0946) or genetic inhibition of DOT1L attenuates the growth of ovarian cancer cells in cell culture and in a mouse xenograft model of ovarian cancer. DOT1L inhibition also resulted in the upregulation of the NKG2D ligand ULBP1 and subsequent increase in natural killer (NK) cell-mediated ovarian cancer eradication. Collectively, our results demonstrate that DOT1L promotes ovarian cancer tumor growth by regulating apoptotic and metabolic pathways as well as NK cell-mediated eradication of ovarian cancer and identifies DOT1L as a new pharmacological target for ovarian cancer therapy.

P1b/2, N=37, Recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2021 --> Jun 2022 | Trial primary completion date: Jun 2021 --> Jun 2022

No abstract available

The DOT1L inhibitor pinometostat in KMT2A-rearranged AML, the menin inhibitors KO-539 and SYNDX-5613 in KMT2Ar and NPM1-mutated AML, and the mutant TP53 inhibitor APR-246 are examples of novel agents targeting specific mutations in AML. AML remains in incurable disease for many patients but advances in genomics, epigenetics, and drug discovery have led to the development of many potential novel therapeutic agents, many of which are being investigated in ongoing clinical trials. Additional studies will be necessary to determine how best to incorporate these novel agents into routine clinical treatment of AML.

Altogether, our findings established Dot1L as a critical regulator of MDSC immunosuppressive function for the first time, and highlighted the therapeutic potential of Dot1L inhibitor for FH treatment.

P1/2, N=1, Completed, National Cancer Institute (NCI) | Active, not recruiting --> Completed | N=36 --> 1 | Trial completion date: Jun 2021 --> Dec 2020 | Trial primary completion date: Jun 2021 --> Dec 2020

Currently, nucleoside DOT1L inhibitors have been developed successfully with the only EPZ5676 entering phase Ⅰ clinical trial in 2013, which was validated as 'orphan drug' toward MLL-rearranged leukemia by FDA. In order to find compounds with better pharmacokinetic properties as DOT1L inhibitors, other types of non-nucleoside skeletons have also been reported successively.

P1/2, N=36, Active, not recruiting, National Cancer Institute (NCI) | Recruiting --> Active, not recruiting

EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process - protein synthesis - that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.

The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

P1/2, N=36, Recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2020 --> Jun 2021 | Trial primary completion date: Jun 2020 --> Jun 2021

P1b/2, N=37, Recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2020 --> Jun 2021 | Trial primary completion date: Jun 2020 --> Jun 2021

The limited efficacy obtained with compound 11 and lack of efficacy observed with subcutaneous administration of EPZ-5676 around the maximally tolerated dose illustrate the difficulty to achieve a sustained level of DOT1L inhibitor in vivo to suppress DOT1L activity sufficiently. Furthermore, H3K79me2 and efficacy in vivo seem disconnected.

This is a first clinical demonstration of the applicability of DOT1L overexpression in gastric tumors.  The work is suggestive of altered proliferation of cells by DOT1L via regulating cyclins and H3K79 methylation. This indicates the role of DOT1L in the prognosis and possible medical intervention of gastric cancer.

Integrative correlative analyses will include genomics, changes in DOT1L-mediated methylation by H3K79 ELISA, and qPCR of HOXA9 and Meis1. PK studies and azacitidine incorporation and DNA methylation studies will also be performed.