Abplatin(IV) Inhibited Tumor Growth On A Patient Derived Cancer Model of Hepatocellular Carcinoma And Its Comparative Multi-Omics Study With Cisplatin

Background: Cisplatin is the most common antitumor alkylating agent of platinum(II) (Pt(II)) in clinic, however it had many side effects. It is necessary to develop low toxicity platinum(IV) (Pt(IV)) drugs. Multi-omics was frequently used to help one understand the mechanism of a certain therapy at the molecular level. Little was known about the mechanism of Pt(IV) drugs, which may be benifical for clinical translation. Methods: We developed a Pt(IV) drug of cisplatin with two hydrophobic aliphatic chains and further encapsulated it with a drug carrier human serum albumin (HSA) to prepare Abplatin(IV). Transcriptomics, metabolomics and lipidomics were performed to clarify the mechanism of Pt(IV) drugs. T-test assay and fold change were used to find the differential substances.Results: We had further shown Abplatin(IV) had better tumor-targeting performance and greater tumor inhibtion rate than cisplatin. Lipidomics study showed that Abplatin(IV) might induce the changes of BEL-7404 cell membrane, and caused the disorder of glycerophospholipids and sphingolipids. In addition, transcriptomics and metabolomics study showed that Abplatin(IV) mainly disturbed more significant purine metabolism pathway than cisplatin.Conclusions: This research highlighted the development of Abplatin(IV) and the use of multi-omics to help one understand the mechanism of action of prodrugs and their DDS, which was the key to the clinical translation of them.

KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

SPDR-2 Histopathological investigation of the oligodendroglial tumors resected following alkylating agent chemotherapy

Oligodendrogliomas, i.e., lower grade gliomas with 1p/19q codeletion, are often responsive to chemotherapy, however, those tumors eventually recur and life-limiting in the majority of patients despite initial chemotherapeutic response. We have been treating those patients with upfront chemotherapy and subsequent resection following tumor volume decrease since 2006. This study aimed to elucidate the histological changes and the mechanism of recurrence after alkylating agent chemotherapy in oligodendrogliomas. Fifteen oligodendrogliomas (Grade 2: 12, Grade 3: 3) resected following tumor volume decrease after alkylating agent chemotherapy were included and compared with their pre-chemotherapy specimens. Histological changes were investigated using hematoxylin-eosin staining, and changes in proliferative activity, status of glioma stem cells (GSCs), and tumor-infiltrating macrophages were assessed using immunohistochemistry. The frequent histological findings following chemotherapy included a sparse glial background, abundant foamy cell infiltration, gliosis, calcification, and nuclear degradation. The Ki-67/MIB-1 index decreased and the number of macrophages increased after chemotherapy. Moreover, the ratio of GSCs to total tumor cells increased after chemotherapy. GSCs and macrophages constitute the mechanism of resistance to and recurrence after alkylating agent chemotherapy in oligodendrogliomas.

Melphalan flufenamide is an Anticancer medication used to treat multiple Myeloma: A Review

Melphalan Flufenamide is a peptide-drug conjugate composed of a peptide conjugated, via an aminopeptidase-targeting linkage, to the alkylating agent melphalan, with potential antineoplastic and anti-angiogenic activities. Upon administration, the highly lipophilic melphalan flufenamide penetrates cell membranes and enters cells. In aminopeptidase-positive tumor cells, melphalan flufenamide is hydrolyzed by peptidases to release the hydrophilic alkylating agent melphalan. This results in the specific release and accumulation of melphalan in aminopeptidase-positive tumor cells. Melphalan alkylates DNA at the N7 position of guanine residues and induces DNA intra- and inter-strand cross-linkages. This results in the inhibition of DNA and RNA synthesis and the induction of apoptosis, thereby inhibiting tumor cell proliferation. Peptidases are overexpressed by certain cancer cells. The administration of melphalan flufenamide allows for enhanced efficacy and reduced toxicity compared to melphalan.1,2,3

Ir-Catalyzed Chemodivergent Allenylic Alkylation of Vinyl Az-ides: Highly Enantioselective Synthesis of α-Allenylic Amides and Ketones

Enantioselective allenylic alkylation reactions of unstabilized enolates have never been reported. We now present a unified fragment-coupling strategy for the first enantioselective synthesis of α-allenylic amides and ketones through allenyl-ic alkylation of vinyl azides. In these chemodivergent reactions, cooperatively catalyzed by Ir(I)/(phosphoramidite,olefin) complex and Sc(OTf)3, vinyl azides act as the surrogate for both amide enolates and ketone enolates. The desiccant (molecular sieves) plays a crucial role in controlling the chemodivergency of this enantioconvergent and regioselective reaction: Under otherwise identical reaction conditions, the presence of the desiccant led to α-allenylic amides while its absence resulted in α-allenylic ketones from the same substrate combinations. Utilizing race-mic allenylic alcohols as the alkylating agent, the overall process represents a dynamic kinetic asymmetric transformation (DyKAT), where both α-allenylic amides and ketones are formed with the same absolute configuration generally with outstanding enantioselectivity. To the best of our knowledge, this is the first example of the use of vinyl azide as the ketone enolate surrogate in an enantioselective transformation.

The Positive Enantiomer of a Novel Chiral DNA Alkylating Agent Exhibits Nanomolar Potency in Hematologic Cancers

LP-184, or (-)-hydroxyurea methylacylfulvene, is a potent DNA alkylating agent that effectively kills solid tumors. It belongs to the acylfulvene compound family known to induce DNA lesions repaired by the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. Here, we show that LP-284, the synthetic positive enantiomer of LP-184, exhibited the greatest and broadest hematologic cancer antiproliferative activities among the 6 acylfulvenes, including illudin S, illudin M, Irofulven (LP-100), the semisynthetic racemic LP-184, the synthetic negative enantiomer LP-184, and LP-284. The distinct pharmacological activities of LP-284 may be due to differences in metabolic activation, transport, or affinity to cellular macromolecules. To determine whether metabolic activation plays a role, we compared the correlation between the expression of Prostaglandin Reductase 1 (PTGR1), the NADPH-dependent oxidoreductase known to convert Irofulven into its active metabolite, and the IC50 of LP-184, Irofulven, and LP-284. We found that the expression level of PTGR1 is highly correlated with LP-184 (r=0.88, p=8.4e-20) and Irofulven (r=0.71, p=4.7e-10) sensitivity, but not with LP-284 (r=-0.01, p=0.93). We also found that the average expression level of PTGR1 is significantly lower in hematologic cancer cell lines (n=180) than in solid tumor cell lines (n=856), indicating the existence of an alternative LP-284 activator in hematologic cells. Next, we checked mutation status, RNA expression, protein expression, and DNA methylation of 489 oxidoreductases, but none of the enzymes was highly correlated with LP-284 activity. To further explore the potential clinical application of LP-284 in hematologic cancers, we conducted cell viability assays in 18 hematologic cancer cell lines and found that LP-284 exhibited nanomolar potency in acute lymphocytic leukemia (average IC50: 351 nM), chronic myeloid leukemia (average IC50: 360 nM), B-cell lymphoma (average IC50: 366 nM), and Multiple Myeloma (MM, IC50: 334 nM). We also investigated the therapeutic potential of LP-284 in combination with spironolactone in treating MM. Spironolactone, an FDA approved drug for hypertension, degrades one of the key TC-NER players ERCC3 in MM, which in turn makes cells more vulnerable to helix-distorting DNA lesions likely caused by LP-284. While Spironolactone alone didn't cause cytotoxicity to the MM cell line RPMI8226, it reduced LP-284 IC50 by 2.4 fold. Taken together, we have demonstrated the importance of stereochemistry in acylfulvene activity. LP-284, likely to be activated through a different route, is a unique and potent acylfulvene for hematologic cancers. Additionally, pharmacological inhibition of the TC-NER pathway greatly promoted LP-284 cytotoxicity. We hypothesize that LP-284 induces DNA lesions, which may be lethal to TC-NER deficient cells and may block transcription of short-lived fusion genes that are essential for cancer cell survival until repaired. Therefore, our discovery of the novel enantiomer LP-284 may provide a targeted therapy option for hematologic cancers with compromised DNA repair. Disclosures Zhou: Lantern Pharma: Current Employment. Biyani: Lantern Pharma: Current Employment. Kathad: Lantern Pharma: Current Employment, Current equity holder in publicly-traded company. Kulkarni: Lantern Pharma: Current Employment. McDermott: Lantern Pharma: Current Employment. Bhatia: Lantern Pharma: Current Employment.

DDRE-29. EGFR INHIBITION DOWNREGULATES MGMT AND SENSITIZES GBM CELLS TO TMZ

Glioblastoma (GBM) is a highly malignant type of adult brain tumor with a poor prognosis. Temozolomide (TMZ), a DNA alkylating agent, has been widely used as an effective first-line chemotherapeutic agent for the treatment of GBM patients. The efficacy of TMZ in GBM depends on the absence of the DNA repair protein MGMT which reverses the DNA damage induced by TMZ. The MGMT promoter is hypermethylated in about 45% of GBMs, resulting in lack of MGMT expression and increased responsiveness to TMZ. TMZ is less effective in MGMT unmethylated GBMs. We propose that EGFR inhibition downregulates MGMT and sensitizes glioma cells to TMZ and a combination of pretreatment with erlotinib followed by TMZ could be a useful therapeutic approach in MGMT expressing GBMs. As our experimental model, we used multiple MGMT unmethylated lines from the Mayo Clinic patient derived xenografts (PDXs) panel. Our data demonstrate that exposure of cells to erlotinib for 48h results in downregulation of MGMT at the mRNA and protein level. Additionally, EGFR inhibition activates the AP-1 transcription factor, and overexpression of AP-1 components Fos and Jun results in decreased MGMT expression in TMZ resistant PDXs, suggesting that AP-1 acts as a transcriptional repressor of MGMT. We further identified that the mice implanted with TMZ resistant PDXs pretreated with afatinib followed by TMZ treatment survived longer compared to those treated with TMZ alone. Thus, the use of EGFR inhibition may enhance the sensitivity of MGMT unmethylated GBMs to TMZ.

EXTH-16. LP-184, A NOVEL ALKYLATING AGENT, IS EFFECTIVE IN GLIOBLASTOMA

Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to prolong survival of patients with newly diagnosed MGMT-methylated GBM. Blood-brain barrier permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT unmethylated GBM) are desperately needed. Lantern Pharma is currently developing LP-184, a hydroxyurea methyl acylfulvene derivative of irofulven guided by its AI platform RADRⓇ analysis showing that tumor cell sensitivity to LP-184 correlates positively with genes commonly upregulated in GBM and either associated with TMZ resistance (e.g. MGMT, UGDH) or tumor promotion (e.g. EGFR, ANXA2). In an in vitro 3D model that mimics the human BBB, the apparent permeability for LP-184 was 1.53E-04 cm/s at 30 minutes, comparable to 1.72E-04 cm/s for TMZ under identical conditions. LP-184 predominantly alkylates DNA at 3’-adenine predicting insensitivity to MGMT expression. In vitro cell toxicity assays in a panel of GBM cell lines and neurospheres reveal an IC50 range of 30 - 400 nM with the MGMT unmethylated cell line LN-18 being among the most sensitive. A single cycle of LP-184 (4 mg/kg, i.v, every other day X 4 doses) induced rapid and near complete regression of subcutaneous U87 xenografts pre-established in immune-deficient mice and statistically significantly prolonged survival of mice bearing pre-established orthotopic U87 xenografts (p < 0.0001). LP-184-induced DNA damage is associated with synthetic lethality in tumor cell lines with decreased expression of Nucleotide Excision Repair (NER) genes. Furthermore, interrogation of clinical databases for expression of 112 NER genes revealed that 25 - 37% of clinical GBM clusters in a low NER expression subgroup, predicting high sensitivity of low NER GBM subsets to LP-184. These findings identify LP-184 as a promising new alkylating agent and support its further development for GBM therapy.