Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Obesity-induced inflammation exacerbates clonal hematopoiesis.

Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis of indeterminate potential (CHIP) is frequent in aging and involves the expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, the risk factors that contribute to CHIP-associated clonal hematopoiesis (CH) are poorly understood. Obesity induces a proinflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data for 47,466 individuals with validated CHIP in the UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in the waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1, and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and that a proinflammatory state could potentiate the progression of CHIP to more significant hematologic neoplasia. The calcium channel blockers nifedipine and SKF-96365, either alone or in combination with metformin, MCC950, or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP-mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in individuals with obesity.

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway.

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/ß or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/ß inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/ß inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.

Potential clinical use of azacitidine and MEK inhibitor combination therapy in PTPN11-mutated juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of childhood. The molecular hallmark of JMML is hyperactivation of the Ras/MAPK pathway with the most common cause being mutations in the gene PTPN11, encoding the protein tyrosine phosphatase SHP2. Current strategies for treating JMML include using the hypomethylating agent, 5-azacitidine (5-Aza) or MEK inhibitors trametinib and PD0325901 (PD-901), but none of these are curative as monotherapy. Utilizing an Shp2E76K/+ murine model of JMML, we show that the combination of 5-Aza and PD-901 modulates several hematologic abnormalities often seen in JMML patients, in part by reducing the burden of leukemic hematopoietic stem and progenitor cells (HSC/Ps). The reduced JMML features in drug-treated mice were associated with a decrease in p-MEK and p-ERK levels in Shp2E76K/+ mice treated with the combination of 5-Aza and PD-901. RNA-sequencing analysis revealed a reduction in several RAS and MAPK signaling-related genes. Additionally, a decrease in the expression of genes associated with inflammation and myeloid leukemia was also observed in Shp2E76K/+ mice treated with the combination of the two drugs. Finally, we report two patients with JMML and PTPN11 mutations treated with 5-Aza, trametinib, and chemotherapy who experienced a clinical response because of the combination treatment.

Novel BH4-BCL-2 Domain Antagonists Induce BCL-2-Mediated Apoptosis in Triple-Negative Breast Cancer.

Targeting the challenging tumors lacking explicit markers and predictors for chemosensitivity is one of the major impediments of the current cancer armamentarium. Triple-negative breast cancer (TNBC) is an aggressive and challenging molecular subtype of breast cancer, which needs astute strategies to achieve clinical success. The pro-survival B-cell lymphoma 2 (BCL-2) overexpression reported in TNBC plays a central role in deterring apoptosis and is a promising target. Here, we propose three novel BH4 mimetic small molecules, SM396, a covalent binder, and two non-covalent binders, i.e., SM216 and SM949, which show high binding affinity (nM) and selectivity, designed by remodeling the existing BCL-2 chemical space. Our mechanistic studies validate the selectivity of the compounds towards cancerous cells and not on normal cells. A series of functional assays illustrated BCL-2-mediated apoptosis in the tumor cells as a potent anti-cancerous mechanism. Moreover, the compounds exhibited efficacious in vivo activity as single agents in the MDA-MB-231 xenograft model (at nanomolar dosage). Overall, these findings depict SM216, SM396, and SM949 as promising leads, pointing to the clinical translation of these compounds in targeting triple-negative breast cancer.

Targeting SHP2 phosphatase in hematological malignancies.

INTRODUCTION: Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) is a ubiquitously expressed, non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene. Gain-of-function (GOF) mutations in PTPN11 are associated with the development of various hematological malignancies and Noonan syndrome with multiple lentigines (NS-ML). Preclinical studies performed with allosteric SHP2 inhibitors and combination treatments of SHP2 inhibitors with inhibitors of downstream regulators (such as MEK, ERK, and PD-1/PD-L1) demonstrate improved antitumor benefits. However, the development of novel SHP2 inhibitors is necessary to improve the therapeutic strategies for hematological malignancies and tackle drug resistance and disease relapse. AREAS COVERED: This review examines the structure of SHP2, its function in various signaling cascades, the consequences of constitutive activation of SHP2 and potential therapeutic strategies to treat SHP2-driven hematological malignancies. EXPERT OPINION: While SHP2 inhibitors have exhibited promise in preclinical trials, numerous challenges remain in translation to the clinic, including drug resistance. Although PROTAC-based SHP2 degraders show better efficacy than SHP2 inhibitors, novel strategies need to be designed to improve SHP2-specific therapies in hematologic malignancies. Genome-wide CRISPR screening should also be used to identify molecules that confer resistance to SHP2 inhibitors. Targeting these molecules together with SHP2 can increase the target specificity and reduce drug resistance.

Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.

Inflammation-induced PELP1 expression promotes tumorigenesis by activating GM-CSF paracrine secretion in the tumor microenvironment.

The inflammatory tumor microenvironment has been implicated as a major player fueling tumor progression and an enabling characteristic of cancer, proline, glutamic acid, and leucine-rich protein 1 (PELP1) is a novel nuclear receptor coregulator that signals across diverse signaling networks, and its expression is altered in several cancers. However, investigations to find the role of PELP1 in inflammation-driven oncogenesis are limited. Molecular studies here, utilizing macrophage cell lines and animal models upon stimulation with lipopolysaccharide (LPS) or necrotic cells, showed that PELP1 is an inflammation-inducible gene. Studies on the PELP1 promoter and its mutant identified potential binding of c-Rel, an NF-κB transcription factor subunit, to PELP1 promoter upon LPS stimulation in macrophages. Recruitment of c-Rel onto the PELP1 promoter was validated by chromatin immunoprecipitation, further confirming LPS mediated PELP1 expression through c-Rel-specific transcriptional regulation. Macrophages that overexpress PELP1 induces granulocyte-macrophage colony-stimulating factor secretion, which mediates cancer progression in a paracrine manner. Results from preclinical studies with normal-inflammatory-tumor progression models demonstrated a progressive increase in the PELP1 expression, supporting this link between inflammation and cancer. In addition, animal studies demonstrated the connection of PELP1 in inflammation-directed cancer progression. Taken together, our findings provide the first report on c-Rel-specific transcriptional regulation of PELP1 in inflammation and possible granulocyte-macrophage colony-stimulating factor-mediated transformation potential of activated macrophages on epithelial cells in the inflammatory tumor microenvironment, reiterating the link between PELP1 and inflammation-induced oncogenesis. Understanding the regulatory mechanisms of PELP1 may help in designing better therapeutics to cure various inflammation-associated malignancies.

Small peptide inhibitor from the sequence of RUNX3 disrupts PAK1-RUNX3 interaction and abrogates its phosphorylation-dependent oncogenic function.

P21 Activated Kinase 1 (PAK1) is an oncogenic serine/threonine kinase known to play a significant role in the regulation of cytoskeleton and cell morphology. Runt-related transcription factor 3 (RUNX3) was initially known for its tumor suppressor function, but recent studies have reported the oncogenic role of RUNX3 in various cancers. Previous findings from our laboratory provided evidence that Threonine 209 phosphorylation of RUNX3 acts as a molecular switch in dictating the tissue-specific dualistic functions of RUNX3 for the first time. Based on these proofs and to explore the translational significance of these findings, we designed a small peptide (RMR) from the protein sequence of RUNX3 flanking the Threonine 209 phosphorylation site. The selection of this specific peptide from multiple possible peptides was based on their binding energies, hydrogen bonding, docking efficiency with the active site of PAK1 and their ability to displace PAK1-RUNX3 interaction in our prediction models. We found that this peptide is stable both in in vitro and in vivo conditions, not toxic to normal cells and inhibits the Threonine 209 phosphorylation in RUNX3 by PAK1. We also tested the efficacy of this peptide to block the RUNX3 Threonine 209 phosphorylation mediated tumorigenic functions in in vitro cell culture models, patient-derived explant (PDE) models and in in vivo tumor xenograft models. These results proved that this peptide has the potential to be developed as an efficient therapeutic molecule for targeting RUNX3 Threonine 209 phosphorylation-dependent tumor phenotypes.

KIBRA connects Hippo signaling and cancer.

The Hippo signaling pathway is a tumor suppressor pathway that plays an important role in tissue homeostasis and organ size control. KIBRA is one of the many upstream regulators of the Hippo pathway. It functions as a tumor suppressor by positively regulating the core Hippo kinase cascade. However, there are accumulating shreds of evidence showing that KIBRA has an oncogenic function, which we speculate may arise from its functions away from the Hippo pathway. In this review, we have attempted to provide an overview of the Hippo signaling with a special emphasis on evidence showing the paradoxical role of KIBRA in cancer.

Sustainable production of camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana.

Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host-plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 µg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.

Clinical Evaluation of P21 Activated Kinase 1 (PAK1) Activation in Gliomas and Its Effect on Cell Proliferation.

Glioblastomas are the primary malignant tumors of brain tissues with poor prognosis and highly invasive phenotypes. Till now Ki-67 LI has emerged as a well-studied proliferation marker that aids in tumor grading, but labeling index alone cannot predict overall survival in gliomas. P21 activated kinase 1 (PAK1) - a serine/threonine kinase has been shown to function as downstream nodule for various oncogenic signaling pathways that promote neoplastic changes. This study is designed to evaluate the expression of PAK1 across various grades and its correlation with Ki-67 LI and overall survival rates among a total number of 140 clinical brain tumors of glioma patients. We also studied the activation status of phospho PAK1 in glioma tissues and established the role of PAK1 in proliferation of glioblatoma cell lines under γ-irradiation.This study provides molecular evidence signifying the role of PAK1 and its activation status in the progression of Gliomas to more aggressive phenotypes.

Current trends and opportunities in targeting p21 activated kinase-1(PAK1) for therapeutic management of breast cancers.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Identifying reliable biomarkers and druggable molecular targets pose to be a significant quest in breast cancer research. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that direct cell motility, cytoskeletal remodelling, and has been shown to function as a downstream regulator for various cancer signalling cascades that promote cell proliferation, apoptosis deregulation and hasten mitotic abnormalities, resulting in tumor formation and progression. The heterogeneity and acquired drug resistance are important factors that challenge the treatment of breast cancer. p21-activated kinase 1 signalling is crucial for activation of the Ras/RAF/MEK/ERK, PI3K/Akt/mTOR and Wnt signalling cascades which regulate cell survival, cell cycle progression, differentiation, and proliferation. A study involving proteogenomics analysis on breast cancer tissues showed the PAK1 as outlier kinase. In addition to this, few outlier molecules were identified specific to subtypes of breast cancer. A few substrates of PAK1 in breast cancer are already known. In this paper, we have discussed a similar approach called Kinase Interacting Substrate Screening (KISS) for the identification of novel oncogenic substrates of p21-activated kinase specific to subtypes of breast cancer. Such high throughput approaches are expected to accelerate the process of identifying novel drug targets and biomarkers.