Targeting RNA editing of antizyme inhibitor 1: A potential oligonucleotide-based antisense therapy for cancer.

Dysregulated adenosine-to-inosine (A-to-I) RNA editing is implicated in various cancers. However, no available RNA editing inhibitors have so far been developed to inhibit cancer-associated RNA editing events. Here, we decipher the RNA secondary structure of antizyme inhibitor 1 (AZIN1), one of the best-studied A-to-I editing targets in cancer, by locating its editing site complementary sequence (ECS) at the 3' end of exon 12. Chemically modified antisense oligonucleotides (ASOs) that target the editing region of AZIN1 caused a substantial exon 11 skipping, whereas ECS-targeting ASOs effectively abolished AZIN1 editing without affecting splicing and translation. We demonstrate that complete 2'-O-methyl (2'-O-Me) sugar ring modification in combination with partial phosphorothioate (PS) backbone modification may be an optimal chemistry for editing inhibition. ASO3.2, which targets the ECS, specifically inhibits cancer cell viability in vitro and tumor incidence and growth in xenograft models. Our results demonstrate that this AZIN1-targeting, ASO-based therapeutics may be applicable to a wide range of tumor types.

Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/ß-catenin-driven hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. METHODS: The HCC tumor model was generated by hydrodynamic transfection of AKT1 and ß-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. RESULTS: Co-activation of both Wnt/ß-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/ß-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/ß-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/ß-catenin-driven HCC. CONCLUSIONS: In conclusion, we have shown that Akt/ß-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/ß-catenin-driven HCC tumors. LAY SUMMARY: The prognosis for patients with hepatocellular carcinoma is poor, partly because of the lack of effective treatment options for those with more advanced disease. In this study, we identified a subpopulation of cancer cells with stem cell-like properties that were critical for tumor maintenance and growth in a mouse model of hepatocellular carcinoma. Through further experiments, we demonstrated that the Jak/Stat pathway is a promising therapeutic target in hepatocellular carcinoma.

Clinical validation of a nanodiamond-embedded thermoplastic biomaterial.

Detonation nanodiamonds (NDs) are promising drug delivery and imaging agents due to their uniquely faceted surfaces with diverse chemical groups, electrostatic properties, and biocompatibility. Based on the potential to harness ND properties to clinically address a broad range of disease indications, this work reports the in-human administration of NDs through the development of ND-embedded gutta percha (NDGP), a thermoplastic biomaterial that addresses reinfection and bone loss following root canal therapy (RCT). RCT served as the first clinical indication for NDs since the procedure sites involved nearby circulation, localized administration, and image-guided treatment progress monitoring, which are analogous to many clinical indications. This randomized, single-blind interventional treatment study evaluated NDGP equivalence with unmodified GP. This progress report assessed one control-arm and three treatment-arm patients. At 3-mo and 6-mo follow-up appointments, no adverse events were observed, and lesion healing was confirmed in the NDGP-treated patients. Therefore, this study is a foundation for the continued clinical translation of NDs and other nanomaterials for a broad spectrum of applications.

Dual-Targeting Dual-Action Platinum(IV) Platform for Enhanced Anticancer Activity and Reduced Nephrotoxicity.

A novel and highly efficient dual-targeting platform was designed to ensure targeted in vivo delivery of dual-action PtIV prodrugs. The dual targeting was established by liposomal encapsulation of PtIV complexes, thereby utilizing the enhanced permeability and retention (EPR) effect as the first stage of targeting to attain a high accumulation of the drug-loaded liposomes in the tumor. After the release of the PtIV prodrug inside cancer cells, a second stage of targeting directed a portion of the PtIV prodrugs to the mitochondria. Upon intracellular reduction, these PtIV prodrugs released two bioactive molecules, acting both on the mitochondrial and on the nuclear DNA. Our PtIV system showed excellent activity in vitro and in vivo, characterized by a cytotoxicity in a low micromolar range and complete tumor remission, respectively. Notably, marked in vivo activity was accompanied by reduced kidney toxicity, highlighting the unique therapeutic potential of our novel dual-targeting dual-action platform.

Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy.

OBJECTIVE: As the current therapeutic strategies for human hepatocellular carcinoma (HCC) have been proven to have limited effectiveness, immunotherapy becomes a compelling way to tackle the disease. We aim to provide humanised mouse (humice) models for the understanding of the interaction between human cancer and immune system, particularly for human-specific drug testing. DESIGN: Patient-derived xenograft tumours are established with type I human leucocyte antigen matched human immune system in NOD-scid Il2rg-/- (NSG) mice. The longitudinal changes of the tumour and immune responses as well as the efficacy of immune checkpoint inhibitors are investigated. RESULTS: Similar to the clinical outcomes, the human immune system in our model is educated by the tumour and exhibits exhaustion phenotypes such as a significant declination of leucocyte numbers, upregulation of exhaustion markers and decreased the production of human proinflammatory cytokines. Notably, cytotoxic immune cells decreased more rapidly compared with other cell types. Tumour infiltrated T cells have much higher expression of exhaustion markers and lower cytokine production compared with peripheral T cells. In addition, tumour-associated macrophages and myeloid-derived suppressor cells are found to be highly enriched in the tumour microenvironment. Interestingly, the tumour also changes gene expression profiles in response to immune responses by upregulating immune checkpoint ligands. Most importantly, in contrast to the NSG model, our model demonstrates both therapeutic and side effects of immune checkpoint inhibitors pembrolizumab and ipilimumab. CONCLUSIONS: Our work provides a model for immune-oncology study and a useful parallel-to-human platform for anti-HCC drug testing, especially immunotherapy.

Epigenetics in cancer stem cells.

Compelling evidence have demonstrated that bulk tumors can arise from a unique subset of cells commonly termed "cancer stem cells" that has been proposed to be a strong driving force of tumorigenesis and a key mechanism of therapeutic resistance. Recent advances in epigenomics have illuminated key mechanisms by which epigenetic regulation contribute to cancer progression. In this review, we present a discussion of how deregulation of various epigenetic pathways can contribute to cancer initiation and tumorigenesis, particularly with respect to maintenance and survival of cancer stem cells. This information, together with several promising clinical and preclinical trials of epigenetic modulating drugs, offer new possibilities for targeting cancer stem cells as well as improving cancer therapy overall.

Nanodiamond-Manganese dual mode MRI contrast agents for enhanced liver tumor detection.

Contrast agent-enhanced magnetic resonance (MR) imaging is critical for the diagnosis and monitoring of a number of diseases, including cancer. Certain clinical applications, including the detection of liver tumors, rely on both T1 and T2-weighted images even though contrast agent-enhanced MR imaging is not always reliable. Thus, there is a need for improved dual mode contrast agents with enhanced sensitivity. We report the development of a nanodiamond-manganese dual mode contrast agent that enhanced both T1 and T2-weighted MR imaging. Conjugation of manganese to nanodiamonds resulted in improved longitudinal and transverse relaxivity efficacy over unmodified MnCl2 as well as clinical contrast agents. Following intravenous administration, nanodiamond-manganese complexes outperformed current clinical contrast agents in an orthotopic liver cancer mouse model while also reducing blood serum concentration of toxic free Mn2+ ions. Thus, nanodiamond-manganese complexes may serve as more effective dual mode MRI contrast agent, particularly in cancer.

Nanodiamond-mitoxantrone complexes enhance drug retention in chemoresistant breast cancer cells.

Chemoresistance is a prevalent issue that accounts for the vast majority of treatment failure outcomes in metastatic cancer. Among the mechanisms of resistance that markedly decrease treatment efficacy, the efflux of drug compounds by ATP-binding cassette (ABC) transporter proteins can impair adequate drug retention by cancer cells required for therapeutic cytotoxic activity. Of note, ABC transporters are capable of effluxing several classes of drugs that are clinical standards, including the anthracyclines such as doxorubicin, as well as anthracenediones such as mitoxantrone. To address this challenge, a spectrum of nanomaterials has been evaluated for improved drug retention and enhanced efficacy. Nanodiamonds (NDs) are emerging as a promising nanomaterial platform because they integrate several important properties into a single agent. These include a uniquely faceted truncated octahedral architecture that enables potent drug binding and dispersibility in water, scalably processed ND particles with uniform diameters of approximately 5 nm, and a demonstrated ability to improve drug tolerance while delaying tumor growth in multiple preclinical models, among others. This work describes a ND-mitoxantrone complex that can be rapidly synthesized and mediates marked improvements in drug efficacy. Comprehensive complex characterization reveals a complex with favorable drug delivery properties that is capable of improving drug retention and efficacy in an MDA-MB-231-luc-D3H2LN (MDA-MB-231) triple negative breast cancer cell line that was lentivirally transduced for resistance against mitoxantrone. Findings from this study support the further evaluation of ND-MTX in preclinical dose escalation and safety studies toward potentially clinical validation.

Synthesis of nanodiamond-daunorubicin conjugates to overcome multidrug chemoresistance in leukemia.

Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains. FROM THE CLINICAL EDITOR: The authors of this study demonstrate superior treatment properties of resistant leukemia cell lines by utilizing nanodiamond vectors loaded with daunorubicin, paving the way to clinical studies in the hopefully not too distant future.

N-of-1 health optimization: Digital monitoring of biomarker dynamics to gamify adherence to metabolic switching.

The digital health field is experiencing substantial growth due to its potential for sustained and longitudinal deployment. In turn, this may drive improved monitoring and intervention as catalysts for behavioral change compared to traditional point-of-care practices. In particular, the increase in incidence of population health challenges such as diabetes, heart disease, fatty liver disease, and other disorders coupled with rising healthcare costs have emphasized the importance of exploring technical, economics, and implementation considerations, among others in the decentralization of health and healthcare innovations. Both healthy individuals and patients stand to benefit from continued technical advances and studies in these domains. To address these points, this study reports a N-of-1 study comprised of sustained regimens of intermittent fasting, fitness (strength and cardiovascular training), and high protein, low carbohydrate diet and parallel monitoring. These regimens were paired with serial blood ketone, blood glucose (wearable and finger stick) and blood pressure readings, as well as body weight measurements using a collection of devices. Collectively this suite of platforms and approaches were used to monitor metabolic switching from glucose to ketones as energy sources-a process associated with potential cardio- and neuroprotective functions. In addition to longitudinal biomarker dynamics, this work discusses user perspectives on the potential role of harnessing digital devices to these dynamics as potential gamification factors, as well as considerations for the role of biomarker monitoring in health regimen development, user stratification, and potentially informing downstream population-scale studies to address metabolic disease, healthy aging and longevity, among other indications.

A functional personalised oncology approach against metastatic colorectal cancer in matched patient derived organoids.

Globally, colorectal cancer (CRC) is the third most frequently occurring cancer. Progression on to an advanced metastatic malignancy (metCRC) is often indicative of poor prognosis, as the 5-year survival rates of patients decline rapidly. Despite the availability of many systemic therapies for the management of metCRC, the long-term efficacies of these regimens are often hindered by the emergence of treatment resistance due to intratumoral and intertumoral heterogeneity. Furthermore, not all systemic therapies have associated biomarkers that can accurately predict patient responses. Hence, a functional personalised oncology (FPO) approach can enable the identification of patient-specific combinatorial vulnerabilities and synergistic combinations as effective treatment strategies. To this end, we established a panel of CRC patient-derived organoids (PDOs) as clinically relevant biological systems, of which three pairs of matched metCRC PDOs were derived from the primary sites (ptCRC) and metastatic lesions (mCRC). Histological and genomic characterisation of these PDOs demonstrated the preservation of histopathological and genetic features found in the parental tumours. Subsequent application of the phenotypic-analytical drug combination interrogation platform, Quadratic Phenotypic Optimisation Platform, in these pairs of PDOs identified patient-specific drug sensitivity profiles to epigenetic-based combination therapies. Most notably, matched PDOs from one patient exhibited differential sensitivity patterns to the rationally designed drug combinations despite being genetically similar. These findings collectively highlight the limitations of current genomic-driven precision medicine in guiding treatment strategies for metCRC patients. Instead, it suggests that epigenomic profiling and application of FPO could complement the identification of novel combinatorial vulnerabilities to target synchronous ptCRC and mCRC.

Pushing the Frontiers of Cancer Research: Highlights from the Frontiers in Cancer Science Conference 2023.

The 15th annual Frontiers in Cancer Science (FCS) conference gathered scientific experts who shared the latest research converging upon several themes of cancer biology. These themes included the dysregulation of metabolism, cell death, and other signaling processes in cancer cells; using patient "omics" datasets and single-cell and spatial approaches to investigate heterogeneity, understand therapy resistance, and identify targets; innovative strategies for inhibiting tumors, including rational drug combinations and improved drug delivery mechanisms; and advances in models that can facilitate screening for cancer vulnerabilities and drug testing. We hope the insights from this meeting will stimulate further progress in the field.

Oncogene-specific formation of chemoresistant murine hepatic cancer stem cells.

UNLABELLED: At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of activation of v-akt murine thymoma viral oncogene homolog (AKT) and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This "side population" (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers, and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ATP binding cassette drug transporter proteins that also contribute to chemoresistance in cancer. We found that the multidrug resistance gene 1 (MDR1) transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1. CONCLUSION: The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer.

Biomedical applications of nanodiamonds: From drug-delivery to diagnostics.

Advances in nanotechnology have great potential to address many unmet clinical and biomedical needs. Nanodiamonds, as a class of carbon nanoparticles with unique properties, may be useful towards a versatile range of biomedical applications from drug delivery to diagnostics. This review describes how these properties of nanodiamonds facilitate their application in different fields of biomedicine, including delivery of chemotherapy drugs, peptides, proteins, nucleic acids and biosensors. Additionally, clinical potential of nanodiamonds, with studies in both preclinical and clinical stages, is also reviewed here, highlighting the translational potential of nanodiamonds in biomedical research.

Splice-switch oligonucleotide-based combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYC-driven hepatocellular carcinoma.

Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC-targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice-switch oligonucleotide (SSO) technologies with our phenotypic-analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target-target interactions and rank synthetic lethality targets. Our SSO-QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC-deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient-derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO-QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC-driven HCC.

NanoBeacon.AI: AI-Enhanced Nanodiamond Biosensor for Automated Sensitivity Prediction to Oxidative Phosphorylation Inhibitors.

Spalt-like transcription factor 4 (SALL4) is an oncofetal protein that has been identified to drive cancer progression in hepatocellular carcinoma (HCC) and hematological malignancies. Furthermore, a high SALL4 expression level is correlated to poor prognosis in these cancers. However, SALL4 lacks well-structured small-molecule binding pockets, making it difficult to design targeted inhibitors. SALL4-induced expression of oxidative phosphorylation (OXPHOS) genes may serve as a therapeutically targetable vulnerability in HCC through OXPHOS inhibition. Because OXPHOS functions through a set of genes with intertumoral heterogeneous expression, identifying therapeutic sensitivity to OXPHOS inhibitors may not rely on a single clear biomarker. Here, we developed a workflow that utilized molecular beacons, nucleic-acid-based, activatable sensors with high specificity to the target mRNA, delivered by nanodiamonds, to establish an artificial intelligence (AI)-assisted platform for rapid evaluation of patient-specific drug sensitivity. Specifically, when the HCC cells were treated with the nanodiamond-medicated OXPHOS biosensor, high sensitivity and specificity of the sensor allowed for improved identification of OXPHOS expression in cells. Assisted by a trained convolutional neural network, drug sensitivity of cells toward an OXPHOS inhibitor, IACS-010759, could be accurately predicted. AI-assisted OXPHOS drug sensitivity assessment could be accomplished within 1 day, enabling rapid and efficient clinical decision support for HCC treatment. The work proposed here serves as a foundation for the patient-based subtype-specific therapeutic research platform and is well suited for precision medicine.

Pazopanib elicits remarkable response in metastatic porocarcinoma: a functional precision medicine approach.

Metastatic porocarcinomas (PCs) are vanishingly rare, highly aggressive skin adnexal tumors with mortality rates exceeding 70%. Their rarity has precluded the understanding of their disease pathogenesis, let alone the conduct of clinical trials to evaluate treatment strategies. There are no effective agents for unresectable PCs. Here, we successfully demonstrate how functional precision medicine was implemented in the clinic for a metastatic PC with no known systemic treatment options. Comprehensive genomic profiling of the tumor specimen did not yield any actionable genomic aberrations. However, ex vivo drug testing predicted pazopanib efficacy, and indeed, administration of pazopanib elicited remarkable clinicoradiological response. Pazopanib and its class of drugs should be evaluated for efficacy in other cases of PC, and the rationale for efficacy should be determined when PC tumor models become available. A functional precision medicine approach could be useful to derive effective treatment options for rare cancers.

Histone-lysine N-methyltransferase EHMT2 (G9a) inhibition mitigates tumorigenicity in Myc-driven liver cancer.

Hepatocellular carcinoma (HCC) is the third deadliest and sixth most common cancer in the world. Histone-lysine N-methyltransferase EHMT2 (also known as G9a) is a histone methyltransferase frequently overexpressed in many cancer types, including HCC. We showed that Myc-driven liver tumours have a unique H3K9 methylation pattern with corresponding G9a overexpression. This phenomenon of increased G9a was further observed in our c-Myc-positive HCC patient-derived xenografts. More importantly, we showed that HCC patients with higher c-Myc and G9a expression levels portend a poorer survival with lower median survival months. We demonstrated that c-Myc interacts with G9a in HCC and cooperates to regulate c-Myc-dependent gene repression. In addition, G9a stabilises c-Myc to promote cancer development, contributing to the growth and invasive capacity in HCC. Furthermore, combination therapy between G9a and synthetic-lethal target of c-Myc, CDK9, demonstrates strong efficacy in patient-derived avatars of Myc-driven HCC. Our work suggests that targeting G9a could prove to be a potential therapeutic avenue for Myc-driven liver cancer. This will increase our understanding of the underlying epigenetic mechanisms of aggressive tumour initiation and lead to improved therapeutic and diagnostic options for Myc-driven hepatic tumours.

Red Blood Cell-Derived Extracellular Vesicles Display Endogenous Antiviral Effects and Enhance the Efficacy of Antiviral Oligonucleotide Therapy.

The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.

Addressing antimicrobial resistance with the IDentif.AI platform: Rapidly optimizing clinically actionable combination therapy regimens against nontuberculous mycobacteria.

Background: Current standard of care (SOC) regimens against nontuberculous mycobacteria (NTM) usually result in unsatisfactory therapeutic responses, primarily due to multi-drug resistance and antibiotic susceptibility-guided therapies. In the midst of rising incidences in NTM infections, strategies to develop NTM-specific treatments have been explored and validated. Methods: To provide an alternative approach to address NTM-specific treatment, IDentif.AI was harnessed to rapidly optimize and design effective combination therapy regimens against Mycobacterium abscessus (M. abscessus), the highly resistant and rapid growth species of NTM. IDentif.AI interrogated the drug interaction space from a pool of 6 antibiotics, and pinpointed multiple clinically actionable drug combinations. IDentif.AI-pinpointed actionable combinations were experimentally validated and their interactions were assessed using Bliss independence model and diagonal measurement of n-way drug interactions. Results: Notably, IDentfi.AI-designed 3- and 4-drug combinations demonstrated greater %Inhibition efficacy than the SOC regimens. The platform also pinpointed two unique drug interactions (Levofloxacin (LVX)/Rifabutin (RFB) and LVX/Meropenem (MEM)) that may serve as the backbone of potential 3- and 4-drug combinations like LVX/MEM/RFB, which exhibited 58.33±4.99 %Inhibition efficacy against M. abscessus. Further analysis of LVX/RFB via Bliss independence model pointed to dose-dependent synergistic interactions in clinically actionable concentrations. Conclusions: IDentif.AI-designed combinations may provide alternative regimen options to current SOC combinations that are often administered with Amikacin, which has been known to induce ototoxicity in patients. Furthermore, IDentif.AI pinpointed 2-drug interactions may also serve as the backbone for the development of other effective 3- and 4-drug combination therapies. The findings in this study suggest that this platform may contribute to NTM-specific drug development.