PIM Inhibitors Research Articles

Genomic Landscape of Primary Cutaneous Diffuse Large B Cell Lymphoma, Leg Type

Primary cutaneous diffuse large B-cell lymphoma, leg type (DLBCL-LT) is an aggressive subtype of cutaneous B cell lymphoma (CBCL) with a poor prognosis. It is most often diagnosed on the legs of the elderly, frequently relapses, and has a tendency towards extracutaneous dissemination. Current treatment strategies are ineffective and revolve around surgical excision, multiagent chemotherapy (e.g. R-CHOP) and radiation therapy, with estimated 5-year survival rates of ~50%. Identifying genetic mutations and altered pathways specific for DLBCL-LT is crucial for the diagnosis and development of effective targeted therapies for this condition.We performed whole exome capture and massively parallel sequencing on 13 tumor samples of DLBCL-LT (4 matched and 9 unmatched) and 5 samples of cutaneous diffuse large B cell lymphoma, not otherwise specified (DLBCL-NOS) (1 matched and 4 unmatched). This is the largest whole exome cohort of cutaneous DLBCL to date. We applied algorithms routinely used by our lab to identify putative cancer promoting mutations and to compare the genetic landscape with DLBCL, both nodal and extranodal.Broadly, the landscape of mutations in DLBCL-LT is similar to that of activated B-cell like (ABC) DLBCL and most analogous to primary central nervous system lymphoma (PCNSL), a type of extranodal ABC DLBCL. Similar to PCNSL, we found frequent disease-defining oncogenic gene mutations in MYD88 (85%), CD79B (38%), and TBL1XR1 (38%), and loss of function mutations in tumor suppressor genes KMT2D (15%), CDKN1B (15%), and CD58 (8%). We observed frequent mutations in PIM1 (54%), OSBPL10 (31%), BTG2 (31%), and IGLL5 (15%), which are either known or proposed targets of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation (aSHM) also seen in PCNSL. Importantly and similarly to PCNSL, we found alterations at the PDL-1 and PDL-2 locus, but instead of copy number gains, we discovered frequent PD-L1 and PD-L2 translocations (40%) that may translate into overexpression of PD-L1 .This spectrum of mutations differed significantly compared to our DLBCL-NOS cohort, which did not have the above oncogenic mutations and lacked any PD-L1 or PD-L2 chromosomal alterations. Finally, we report novel oncogenic mutations in DLBCL-LT including MYD88 p.S243N, CD79B p.L199P, CCND3 p.T283P and p.P284S, BRAF p.V600E, and CARD11 p.R113Q and novel loss of function mutations in KMT2D, CDKN1B, CD58, MSH2, PIK3R1, MSH2, and BMF .Collectively, we identified recurrent mutations in multiple key cancer-related biological pathways, including epigenetic modulators (TBL1XR1, HIST1H1E, and KMT2D), NF-κB signaling pathway (MYD88, CD79B, and CARD11), MAPK signaling pathway (BRAF), PI3K signaling pathway (PIK3R1), JAK/STAT signaling pathway (PIM1), cell cycle regulators (CCND3, CDKN1B and BTG2), and immune escape (CD58). All of the DLBCL-LT samples in our cohort contained one or more gene mutations that can be targeted by agents that are currently FDA-approved or under clinical trials, such as BRAF inhibitors, JAK/STAT inhibitors, PIM inhibitors, PI3K inhibitors, NF-kB inhibitors, MYD88 inhibitors, and PD-1/PDL-1 inhibitors.Altogether, our data identified targetable putative driver gene mutations in DLBCL-LT. These mutational signatures bear broad similarities to PCNSL. Because of the relative rarity of this disease, we hope these efforts will allow for its inclusion, when relevant, in trials involving genotypically similar DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-Myeloma Activity Via Cereblon-IKZF1/3 Cascade

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-myeloma Activity Via Cereblon-IKZF1/3 CascadeYonggang Sha1, Jing Zheng2, Jianda Hu2+, and Yubin Kang1+1Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA; 2Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, China+ Corresponding author: Yubin Kang, MD, Email: yubin.kang@duke.edu; and Jianda Hu, Email: drjiandahu@163.com.ABSTRACTPurpose: Multiple myeloma (MM) remains an incurable disease, and continuous efforts are required to develop novel therapeutic agents and novel drug combinations with more effective anti-myeloma activity. Immunomodulatory agents (IMiDs) including lenalidomide are one of the mainstays in the treatment of MM. It is important to identify and develop novel agents that can work with IMiDs for enhanced anti-myeloma effects. Here, we determined anti-myeloma effect of pan-PIM inhibitors and the synergistic anti-myeloma effects of pan-PIM inhibitors with IMiDs (lenalidomide).Experimental Design: The anti-myeloma activities of pan-PIM inhibitors (SGI1776 and CX6258) and the synergistic anti-myeloma effects of pan-PIM inhibitors with lenalidomide were determined in human myeloma cells, transplanted VK*MYC myeloma mice, and myeloma xenograft mouse models. Western blot, protein stability assay, shRNA specific gene knockdown, and ubiquitination assay were performed to determine the molecular mechanisms involved.Results: Pan-PIM inhibitors (SGI1776 and CX6258) induced the apoptosis of MM cells and exhibited significant anti-myeloma activities in human myeloma cells and in transplanted VK*MYC myeloma mice. Combination of PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities in vivo in myeloma xenograft mouse models. The treatments of pan-PIM inhibitors promoted ubiquitination and degradation of IKZF1 and IKZF3, the two transcription factors important for the survival of myeloma cells, and combining Pan-PIM inhibitors with lenalidomide leds to more effective degradation of IKZF1 and IKZF3 in vitro in human multiple myeloma cells and in vivo in myeloma xenograft mouse model. We further demonstrated that treatments with pan-PIM inhibitors resulted in an increased expression of Cereblon, and knockdown of Cereblon with shRNA abolished the effects of pan-PIM inhibitors on IKZF1 and IKZF3, demonstrating a central role of Cereblon in Pan-PIM inhibitor-mediated IKZF1/3 degradation.Conclusions: We demonstrated that pan-PIM inhibitors (SGI1776 and CX6258) exhibited significant anti-myeloma activities and combination of pan-PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities. Mechanistically, pan-PIM kinase inhibitors enhance lenalidomide's anti-myeloma activity via Cereblon-IKZF1/3 Cascade. Our study provides molecular rationale and justification for clinical trials combining pan-PIM inhibitors and lenalidomide for the treatment of myeloma. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Pim-2 kinase by LT-171-861 promotes DNA damage and exhibits enhanced lethal effects with PARP inhibitor in multiple myeloma

Multiple myeloma (MM) is a malignancy of antibody-producing plasma cells with genomic instability and genetic abnormality as its two hallmarks. Therefore, DNA damage is pervasive in MM cells, which indicates irregular DNA damage response (DDR) pathway. In this study, we demonstrated that LT-171-861, a multiple kinase inhibitor, could inhibit proliferation and induce apoptosis in MM cells. LT-171-861 promoted DDR pathway and triggered DNA damage through impeding the process of homologous recombination in double strand breaks, rather than directly elevating ROS level in MM cells. Mechanism research revealed that Pim2 inhibition was responsible for LT-171-861-indcued DNA damage and cell apoptosis. LT-171-861 mainly suppressed Pim2 kinase activity and reduced the expression of its phosphorylated substrates, such as 4EBP1 and BAD. Moreover, Olaparib, a PARP inhibitor, could enhance the antitumor effect of LT-171-861 in suppressing tumor growth in MM xenografted nude mice. Taken together, our results demonstrated that LT-171-861 showed a promising therapeutic potential for MM and had an additional lethal effect with PARP inhibitors.

Updated results from DIAMOND-01 (CLI24-001) trial: A phase I/II study of SEL24/MEN1703, a first-in-class dual PIM/FLT3 kinase inhibitor, in acute myeloid leukemia.

7023 Background: SEL24/MEN1703, a dual PIM/FLT3 kinase inhibitor, in the dose escalation (DE) DIAMOND-01 trial (CLI24-001, NCT03008187), showed an acceptable safety profile up to the recommended dose (RD) of 125 mg along with initial evidence of single agent activity and meaningful target engagement in heavily pre-treated patients (pts) with AML (Solomon et al, EHA 2020; Tomirotti et al, ASH 2020). Here we present updated data including pts enrolled in the Phase II, cohort expansion (CE) of the study. Methods: DIAMOND-01 trial enrolled pts unsuitable for chemotherapy having relapsed or refractory (R/R) (DE and CE) or previously untreated (DE) AML. Previous targeted therapies – except PIM inhibitors – were allowed. SEL24/MEN1703 was given orally, QD, 14 days ON / 7 days OFF until progression/unacceptable toxicity. The DE tested MEN1703 escalating doses from 25 to 150 mg, whereas in the CE the RP2D (125 mg) was administered. The key objectives of the CE were the confirmation of the safety profile determined in the DE along with further investigation of single agent activity. Adverse events (AEs) were graded according to NCI-CTCAE v.4.03; responses assessed as per ELN 2017 criteria. Results: As of January 21, 2021 (cut-off date), n = 48 pts were treated across DE (n = 25) and CE (n = 23). Median age was 69 (25-84) years. Overall, 20 (43%) and 15 (32%) pts had non de novo AML and primary refractory AML, respectively. Adverse karyotype was reported in 7 (15%) pts. Most frequently reported mutations were FLT3/ITD (23%, n = 11), DNMT3A (15% n = 7), NPM1 (15%, n = 7), IDH1 (13%, n = 6) and IDH2 (4%, n = 2), CEBPA (4%, n = 2), FLT3/TKD (2%, n = 1). Median number of cycles was 2 (1-8). At the RD (n = 30), most frequent serious treatment-emergent AEs (serious TEAEs) were pneumonia (23%), sepsis and febrile neutropenia (13%) and pulmonary mycosis (10%) whereas most frequent G≥3 TEAEs were febrile neutropenia and pneumonia (23%), leukocytosis (20%) and neutrophil count decrease, platelet count decrease, lymphocyte count decrease and sepsis (13%). Responses occurred in 2 pts in the CE, both with IDH1 mutant disease (naïve to IDH inhibitors) who achieved complete remission with incomplete hematologic recovery (CRi). Both responses occurred by Cycle 3, with a duration of 79 (ongoing at cut-off date) and 43 days, respectively. Across DE and CE, 4 CR/CRi occurred, three of which in pts with IDH mutations. A total of 3 out of 6 pts with IDH mutations treated at doses ≥75 mg achieved CR/CRi, including a CR in a patient with IDH2 mutant AML relapsed on Enasidenib. Conclusions: SEL24/MEN1703 confirmed a manageable safety profile at RD and showed preliminary single agent efficacy in R/R AML, particularly clustering in pts with IDH mutant disease either naïve or previously exposed to IDH inhibitors. These results warrant further investigation of SEL24/MEN1703 in AML, with potential focus in the IDH mutated subset. Clinical trial information: NCT03008187.

Synthesis of new substituted pyridine derivatives as potent anti-liver cancer agents through apoptosis induction: In vitro, in vivo, and in silico integrated approaches

Liver cancer is the most common type of cancer in many countries. New studies and statistics show rising liver cancer worldwide, so it is essential to seek new agents for this type of cancer. PIM1 has an attractive target in the discovery of cancer medications as it is very much expressed in a variety of malignancies and influences such as tumorigenesis, cell cycle progression, cellular proliferation, apoptosis, and cell migration. Accordingly, a series of pyridones and pyridine-amides were synthesized and tested for anti-liver cancer activity.In the synthetic strategy 4,6-diaryl-3-cyano-2-pyridones 3a-n were synthesized using one-pot four component synthetic method. Structural modifications were done on 4,6-diphenyl-3-cayno-2-pyridone 3a to enhance the activity. Alkylation in the presence of K2CO3 afforded the O-alkylated products 4–6. The acetoxy hydrazide 7 was synthesized and cyclized into 1,3,4-oxadiazolethione 8 which alkylated on sulfur to give 10. Azide-coupling method was used to couple the 2-(pyridin-2-yloxy)acetohydrazide 7 to different amines and amino acid esters to furnish the products 12a-e and 13a-b. The synthesized derivatives were subjected to cytotoxic screening against HepG2 and THLE-2 cells, Compounds 10, 12e and 13a have a remarkable cytotoxic activity with IC50 values (10.7–13.9 µM). Compound 7 was found to be more cytotoxic by showing the lowest IC50 value of 7.26 compared to 5-FU (IC50 = 6.98 µM). It inhibited cell growth by 76.76%. Additionally, it significantly stimulated apoptotic liver cancer cell death with 49.78-fold (22.90% compared to 0.46% for the control) arresting cell cycle Pre-G1 with 35.16% of a cell population, compared to 1.57% for the control. Moreover, it validated the intrinsic apoptosis through upregulation of P53, and other related genes, with inhibition of anti-apoptotic genes through PIM-1 inhibition.

Structure-Based Virtual Screening and De Novo Design of PIM1 Inhibitors with Anticancer Activity from Natural Products.

Background: the proviral insertion site of Moloney murine leukemia (PIM) 1 kinase has served as a therapeutic target for various human cancers due to the enhancement of cell proliferation and the inhibition of apoptosis. Methods: to identify effective PIM1 kinase inhibitors, structure-based virtual screening of natural products of plant origin and de novo design were carried out using the protein–ligand binding free energy function improved by introducing an adequate dehydration energy term. Results: as a consequence of subsequent enzyme inhibition assays, four classes of PIM1 kinase inhibitors were discovered, with the biochemical potency ranging from low-micromolar to sub-micromolar levels. The results of extensive docking simulations showed that the inhibitory activity stemmed from the formation of multiple hydrogen bonds in combination with hydrophobic interactions in the ATP-binding site. Optimization of the biochemical potency by chemical modifications of the 2-benzylidenebenzofuran-3(2H)-one scaffold led to the discovery of several nanomolar inhibitors with antiproliferative activities against human breast cancer cell lines. Conclusions: these new PIM1 kinase inhibitors are anticipated to serve as a new starting point for the development of anticancer medicine.

PIM kinases mediate resistance to cisplatin chemotherapy in hepatoblastoma

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54–80% of patients developing resistance to chemotherapy after 4–5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

Design and synthesis of a novel mitochondria-targeted osteosarcoma theranostic agent based on a PIM1 kinase inhibitor

Osteosarcoma (OS) is the most common malignancy of the skeletal system, with a poor prognosis and high rate of recurrence. Adequate surgical margin and adjuvant chemotherapy improve the overall survival and limb salvage rate of osteosarcoma patients. Previous studies have showed that OS exhibits an increase in the expression of proviral integration site for Moloney murine leukemia virus 1 (PIM1) kinase, and high levels of PIM1 are also associated with poor OS prognosis and metastasis. We exploited the overexpression of proto-oncogenic PIM1 in OS towards the development of a novel near-infrared imaging and targeted therapeutic agent, namely QCAi-Cy7d by conjugating a PIM1 small molecule inhibitor and heptamethine cyanine dye, for simultaneous guiding surgery and chemotherapy. QCAi-Cy7d showed targeted imaging and anticancer activities against OS in vitro and vivo without any obvious toxicity, and its antitumoral activity was much greater than the parent PIMI inhibitor. These results demonstrated the potential of new conjugate of PIM1 inhibitor and near-infrared imaging, supporting structure-based design and development of theranostic agents for precise tumor imaging and targeted treatment.

PIM447 inhibits oncogenesis and potentiates cisplatin effects in hepatoblastoma

Background: Novel therapies are needed for patients with hepatoblastoma because of an increasing incidence of disease and poor prognosis for advanced, refractory, and recurrent disease. PIM kinases promote tumorigenesis in hepatoblastoma. A novel PIM inhibitor, PIM447, has shown promise in inhibiting oncogenesis in hematologic and lymphoid malignancies. We hypothesized that PIM inhibition with PIM447 would result in decreased tumorigenesis in hepatoblastoma. Methods: The effects of PIM447 on hepatoblastoma viability, proliferation, motility, apoptosis, and tumor cell stemness were assessed in HuH6, a human hepatoblastoma cell line, and COA67, a human hepatoblastoma patient-derived xenograft. Results: PIM447 significantly decreased the viability, proliferation, and motility of HuH6 and COA67 cells. Apoptosis significantly increased following PIM447 treatment. PIM447 had a significant impact on tumor cell stemness as evidenced by decreased expression of CD133 and reduced ability of HuH6 and COA67 cells to form tumorspheres. Furthermore, combining PIM447 with cisplatin resulted in a significant decrease in cell viability compared to either treatment alone. Conclusion: We showed that PIM447 inhibits oncogenesis and potentiates the effects of cisplatin in hepatoblastoma and, therefore, warrants further investigation as a potential therapeutic agent for hepatoblastoma.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Targeting PIM Kinases to Overcome Therapeutic Resistance in Cancer.

Cancer progression and the onset of therapeutic resistance are often the results of uncontrolled activation of survival kinases. The proviral integration for the Moloney murine leukemia virus (PIM) kinases are oncogenic serine/threonine kinases that regulate tumorigenesis by phosphorylating a wide range of substrates that control cellular metabolism, proliferation, and survival. Because of their broad impact on cellular processes that facilitate progression and metastasis in many cancer types, it has become clear that the activation of PIM kinases is a significant driver of resistance to various types of anticancer therapies. As a result, efforts to target PIM kinases for anticancer therapy have intensified in recent years. Clinical and preclinical studies indicate that pharmacologic inhibition of PIM has the potential to significantly improve the efficacy of standard and targeted therapies. This review focuses on the signaling pathways through which PIM kinases promote cancer progression and resistance to therapy, as well as highlights biological contexts and promising strategies to exploit PIM as a therapeutic target in cancer.

Role of oncogene PIM-1 in the development and progression of papillary thyroid carcinoma: Involvement of oxidative stress

In this study, we aimed to clarify the role of PIM-1 in papillary thyroid carcinoma (PTC) in vitro and investigate the relationship between PIM-1 and redox proteins (NOX4, SOD2, and GPX2) at the tissue and cellular levels. As a PIM-1 inhibitor, SGI-1776 inhibited cell proliferation, colony formation, migration and induced an increase in apoptosis and reactive oxygen species in two PTC cell lines (BCPAP and TPC-1). The expressions of PIM-1, SOD2 and GPX2 were downregulated after siNOX4 exposure. Immunohistochemistry in 120 PTC patients showed that all four proteins exhibited higher expression levels in PTC tissues than in adjacent normal tissues. PIM-1 expression was related to NOX4, SOD2, and GPX2 expressions. The Cancer Genome Atlas database analysis showed the significant correlation between the expression of NOX4 and PIM-1. Our results demonstrated that PIM-1 played an important oncogenic role in PTC carcinogenesis that may be related to oxidative stress.

PIM Kinases Promote Survival and Immune Escape in Primary Mediastinal Large B-Cell Lymphoma through Modulation of JAK-STAT and NF-κB Activity

Primary mediastinal large B-cell lymphoma (PMBL) cells depend on the constitutive activity of NF-κB and STAT transcription factors, which drive expression of multiple molecules essential for their survival. In a molecularly related B-cell malignant tumor (classic Hodgkin lymphoma), tumor Reed-Sternberg cells overexpress oncogenic (proviral integration site for Moloney murine leukemia virus (PIM) 1, 2, and 3 kinases in a NF-κB- and STAT-dependent manner and PIMs enhance survival and expression of immunomodulatory molecules. Given the multiple overlapping characteristics of Reed-Sternberg and PMBL cells, we hypothesized that PIM kinases may be overexpressed in PMBL and involved in PMBL pathogenesis. The expression of PIM kinases in PMBL diagnostic biopsy specimens was assessed and their role in survival and immune escape of the tumor cells was determined. PIMs were abundantly expressed in primary tumors and PMBL cell lines. Inhibition of PIM kinases was toxic to PMBL cells, attenuated protein translation, and down-regulated NF-κB- and STAT-dependent transcription of prosurvival factors BCL2A1, BCL2L1, and FCER2. Furthermore, PIM inhibition decreased expression of molecules engaged in shaping the immunosuppressive microenvironment, including programmed death ligand 1/2 and chemokine (C-C motif) ligand 17. Taken together, our data indicate that PIMs support PMBL cell survival and immune escape and identify PIMs as promising therapeutic targets for PMBL.

Synergistic role of JAK/STAT5 and PI3K/AKT signaling pathways in regulating eIF4B in acute leukemia

Human acute leukemia (AL) is a clonal malignancy with abnormal hematopoietic stem cells. Clinically, AL is very difficult to cure due to its sudden onset and short course of disease progression. Previous studies have shown that eukaryotic initiation factor 4B (eIF4B) plays a critical role in the development of chronic leukemia. However, the involvement of eIF4B in human acute leukemia is still largely unknown. Therefore, we studied eIF4B function and its regulatory mechanism in human acute leukemia. We found that phosphorylation levels of eIF4B in acute leukemia cells were significantly reduced in response to treatment with either LY294002 (PI3K inhibitor), AKTi (AKT inhibitor) or SMI-4A (Pim inhibitor). Co-treatment with inhibitors targeting JAK/STAT5/Pim and PI3K/AKT/mTOR signaling dramatically promoted apoptosis of acute leukemia cells by downregulating eIF4B phosphorylation. Furthermore, in vitro and in vivo functional experiments showed that eIF4B played an important anti-apoptosis role in the acute leukemia cells by regulating the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL. In contrast, silencing eIF4B inhibited the growth of acute leukemia cells as engrafted tumors in nude mice. Taken together, our results indicate the synergistic role of JAK/STAT5/Pim and PI3K/AKT/mTOR signaling pathways in regulating eIF4B phosphorylation in acute leukemia, and highlight eIF4B as a candidate therapeutic target for treatment of acute leukemia.

PIM1 inhibition attenuated endotoxin-induced acute lung injury through modulating ELK3/ICAM1 axis on pulmonary microvascular endothelial cells.

The dysfunction of pulmonary microvascular endothelial cells (PMVECs) is one of the critical characteristics of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) induced by severe infection. PIM1 is a constitutively active serine/threonine kinase that is involved in multiple biological processes. However, the underlying correlation between PIM1 and PMVECs injury remains unclear. The main purpose of this study was to reveal roles of PIM1 and explore the potential mechanisms during the development of endotoxin-induced ALI induced by intraperitoneal LPS administration. PIM1 level in the lung tissues of endotoxin-induced ALI mice or plasma derived from cardiopulmonary bypass (CPB)-induced ALI patients were measured. The protective roles of PIM1 specific inhibitor SMI-4a on endotoxin-induced lung injuries were evaluated through histological, permeability, neutrophil infiltration and survival assessment. The relationship between PIM1 and ELK3/ICAM-1 axis was validated in vivo and vitro. The correlation between plasma PIM1 and indicative vascular endothelium injury biomarkers (PaO2/FiO2 ratio, Ang-II, E-selectin and PAI-1) levels derived from CPB-induced ALI patient were analyzed. PIM1 expression in the lung tissues was increased in the mice of endotoxin-induced ALI. The PIM1 specific inhibitor SMI-4a administration relieved the severity of endotoxin-induced ALI. More importantly, PIM1 modulates ICAM1 expression through regulating transcription factor ELK3 expression in vitro. Eventually, plasma PIM1 level was positively correlated with Ang-II and PAI-1 levels but negatively correlated with SpO2/FiO2 ratio among CPB induced ALI patients. Our results indicated that PIM1 inhibition carried a protective role against endotoxin-induced ALI by modulating the ELK3/ICAM1 axis on PMVECs. PIM1 may be a potential therapeutic target for endotoxin-induced ALI.

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

PIM Kinase Inhibition Decreases the Proangiogenic Properties of Multiple Myeloma Cells and Affects the Metabolic State of the Vascular Endothelium

The development and progression of multiple myeloma (MM) depend on the formation and perpetual evolution of an immunosuppressive and hypervascular bone marrow microenvironment. MM undergoes an angiogenic switch during its early progression stages and initiates the secretion of proangiogenic proteins, such as VEGFA and Galectin-1. Following their engagement with the VEGF receptor 2 on the surface of the endothelium, quiescent endothelial cells (ECs) rapidly switch to an activated state, thus gaining the ability to create sprouts, migrate and proliferate. However, chronic angiogenic stimulation results in the formation of a dense and leaky network of pathological vessels, which in the case of MM also serves as a major source of prosurvival paracrine signals. Since PIM kinases are known modulators of cytokine signaling, owing to their ability to activate NFκB, JAK/STAT and mTOR pathways, we analyzed the expression pattern of PIM1, PIM2 and PIM3 in multiple myeloma bone marrow samples using immunohistochemistry. We found that both MM cells as well as myeloma-associated ECs exhibit a significantly higher PIM3 expression than their normal bone marrow counterparts. Since the role of PIM kinases in the vascular compartment of the tumor microenvironment is currently unknown, we decided to explore the proangiogenic functions of PIM kinases using in vitro MM and EC model cell lines. 3 MM cell lines (RPMI 8226, MM1.s, U266), immortalized bone marrow ECs (HBMEC-60) and human umbilical vein ECs (HUVECs) were used for the experiments. Primary MM cells were obtained from MACS-separated bone marrow aspirates. Chemical blockade of PIM kinase activity was achieved using the pan-PIM inhibitor SEL24/MEN1703. The compound decreased the viability of MM cell lines with IC50 in the submicromolar range, induced G2 cell cycle arrest and apoptosis. Moreover, SEL24/MEN1703 induced apoptosis in primary MM cells, even when cocultured with the CD138- bone marrow fraction. PIM inhibitor treatment inhibited the phosphorylation of mTOR substrates S6 and 4EBP1, STAT3/5, as well as RelA/p65. Consequently, we observed markedly decreased VEGFA and Gal-1 levels in SEL24/MEN1703-treated MM cells. When cultured together, separated by a permeable transwell membrane, both RPMI 8226 cells, as well as ECs, exhibited a 2-fold increase in proliferation rate. This effect was completely blocked by a 2-day treatment with a PIM inhibitor. Exposure of ECs to recombinant VEGFA (10ng/ul) or MM supernatant resulted in an increase in VEGFR2 Y1175 phosphorylation level and induction of PIM3 expression. Increased MYC activity is a hallmark of VEGF-dependent endothelial activation and is necessary to support the creation of new vessels. Since the PIM3 promoter region contains putative MYC-binding sites (E-boxes), we checked if PIM3 induction depends on MYC in ECs. MYC silencing using siRNA resulted in an 88% lower PIM3 expression than the non-targeting siRNA. One of MYC's main tasks during angiogenesis is the stimulation of cellular ATP synthesis to meet the energy demands created by the dynamic remodeling of the actin cytoskeleton. Surprisingly, PIM inhibition decreased the total ATP content in ECs by 25%, thus disrupting the energetic homeostasis, as evidenced by a 9.6-fold increase in phosphorylated AMPK T172 levels. Furthermore, SEL24/MEN1703-treated ECs were depleted of higher-order actin structures necessary for efficient angiogenesis, such as actin stress fibers, membrane ruffles and lamellipodia. In consequence, PIM kinase inhibition decreased proliferation, migration and formation of new vessel-like structures in Matrigel by ECs. Collectively, our data demonstrate that PIM inhibition induces MM cell death and abolishes important tumor cell-ECs interactions. In addition, we show that PIM3 is overexpressed in MM tumor endothelial cells and PIM inhibition disrupts the activation state in in vitro cultured ECs. Hence, targeting PIM kinases may represent an efficient approach to induce tumor cell death and to block angiogenesis in MM. RNA-sequencing studies on the downstream effectors of PIM3 are currently ongoing in order to unravel the molecular mechanism behind the observed effects. Figure Disclosures Brzózka: Ryvu Therapeutics: Current Employment. Rzymski:Ryvu Therapeutics: Current Employment. Tomirotti:Menarini Ricerche: Current Employment. Lech-Marańda:Roche, Novartis, Takeda, Janssen-Cilag, Amgen, Gilead, AbbVie, Sanofi: Consultancy; Roche, Amgen, Gilead: Speakers Bureau. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

In silico studies on pyrazine derivatives for identifying potential inhibitors of PIM-1 kinase

PIM-1 kinase (PIM-1K) modulates multiple cellular functions and is evolving as a drug target for cancer. In search of potential PIM-1 inhibitors, we report herein the 3D-QSAR as well as docking studies on 3-(pyrazin-2-yl)-1H-indazole derivatives. Based on the 3D-QSAR study, the generated pharmacophore was utilized for the virtual screening of thousands of compounds (compds) from ZINC database against PIM-1K. Four top-ranked compds, ZINC05885218, ZINC05888770, ZINC08652441, and ZINC73096248, were selected by virtual screening study. The study results of molecular docking suggested that certain key residues were significant for interactions of ligand–receptor due to the formation of hydrogen bonds with Glu171, Glu121, Lys67, Asp128, Asp131 and Asp186 of PIM-1K. Virtually screened compds displayed resemblance in binding interactions within the PIM-1Ks catalytic pocket in comparison with their corresponding crystal structures. The ADRRR.2 emerged as the potential pharmacophore hypothesis and was employed for the generation of model of 3D-QSAR which gave good statistical values of q2 and r2 using partial least square analyses (r2 = 0.922, q2 = 0.8629) for the best model. The study results revealed that ZINC73096248 could serve as a prototype compd for developing novel PIM-1K inhibitors.

Discovery of novel pyrazolo[3,4-b]pyridine scaffold-based derivatives as potential PIM-1 kinase inhibitors in breast cancer MCF-7 cells

Pim-1 kinase targeted recently has proved an essential goal of breast cancer therapy. We report the design, synthesis with full characterization analysis of pyrazolo[3,4-b]pyridine scaffold-based derivatives targeting Pim-1 kinase as anti-breast cancer agents. All the newly synthesized compounds were screened for their in vitro cytotoxic activity against two breast cancer cell lines MCF-7 and MDA-MB-231, and non-cancerous MCF-10A cells. Four derivatives notably, 17 and 19 exhibited a remarkable cytotoxic activity with IC50 values 5.98 and 5.61 µM against MCF-7 (ERα-dependent) cells in a selective way, as they weren’t active against MDA-MB-231 (non-ERα-dependent) and safe against MCF-10A. The most active compounds through in vitro screening were subjected to PIM-1 kinase to elucidate the Pim-1 kinase inhibitory activity as the mechanistic mode of action. Among the tested derivatives, Compounds 17 and 19 showed the highest inhibitory activity with IC50 values 43 and 26 nM, respectively, compared to the 5-FU with IC50 value 17 nM. Moreover, apoptotic investigation through flow cytometry and gene expression analysis of the apoptosis-related genes for the most active compound 19 against MCF-7. It was found that compound 19 induced apoptotic MCF-7 cell death by cell cycle arrest at G2/M phase and by elevation the expression of pro-apoptotic genes and inhibition of anti-apoptotic genes expression. Finally, the PIM-1 inhibition activities for compounds 17 and 19 were in accordance with the molecular docking study that revealed good interaction with the Pim-1 kinase active site.

New cyanopyridine-based scaffold as PIM-1 inhibitors and apoptotic inducers: Synthesis and SARs study

Two novel series of 6-(4-benzamido-/4-phthalimido)-3-cyanopyridine derivatives were designed and synthesized as inhibitors of PIM-1 kinase. Based on cytotoxicity results via MTT assay against prostate carcinoma PC3, human hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cell lines, the most potent cytotoxic cyanopyridine hits, 6, 7, 8, 12 and 13 were 1.5–3.3 times more inhibitor of cell proliferation than the reference standard, 5-FU. Selectivity profile of the latter compounds on normal human cells (WI-38), was executed, indicating that they are highly selective (IC50 > 145 μM) in their cytotoxic effect. The promising compounds were further evaluated as PIM-1 kinase inhibitors. These compounds elicited remarkable inhibition of PIM-1 kinase (76.43–53.33%). Extensive studies on apoptosis were conducted for these compounds; they enhanced caspase-3 and boosted the Bax/Bcl-2 ratio 27-folds in comparison to the control. Molecular docking study of the most potent compound, 13 in PIM-1 kinase active site was consistent with the in vitro activity. Finally, prediction of chemo-informatic properties released compound 13 as the most promising ligand.