PIM Inhibitors Research Articles

Targeting cytokine- and therapy-induced PIM1 activation in preclinical models of T-cell acute lymphoblastic leukemia and lymphoma

AbstractT-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non–cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non–cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.

PAI1 is a Marker of Bad Prognosis in Rectal Cancer but Predicts a Better Response to Treatment with PIM Inhibitor AZD1208.

Colorectal cancer (CRC) is the third most common cancer worldwide. The standard treatment in locally advanced rectal cancer is preoperative radiation alone or in combination with chemotherapy, followed by adjuvant chemotherapy. Rectal cancer is highly lethal, with only 20% of patients showing a complete remission (by RECIST) after standard treatment, although they commonly show local or systemic relapse likely due to its late detection and high chemotherapy resistance, among other reasons. Here, we explored the role of PAI1 (Serpin E1) in rectal cancer through the analyses of public patient databases, our own cohort of locally advanced rectal cancer patients and a panel of CRC cell lines. We showed that PAI1 expression is upregulated in rectal tumors, which is associated with decreased overall survival and increased metastasis and invasion in advanced rectal tumors. Accordingly, PAI1 expression is correlated with the expression of (Epithelial-to-Mesenchymal Transition) EMT-associated genes and genes encoding drug targets, including the tyrosine kinases PDGFRb, PDGFRa and FYN, the serine/threonine kinase PIM1 and BRAF. In addition, we demonstrate that cells expressing PAI1 protein are more sensitive to the PIM inhibitor AZD1208, suggesting that PAI1 could be used to predict response to treatment with PIM inhibitors and to complement radiotherapy in rectal tumors.

Synthesis of Thiazole, Thiophene, Pyran and Pyridine Derivatives Derived from 3-phenyl-1H-pyrazol-5(4H)-one with Anti-proliferative, Tyrosine Kinase and PIM-1 Kinase Inhibitions

Background: A wide range of pyrazole derivatives gained special attention due to their wide range of pharmacological activities especially the therapeutic activities. Many pharmacological drugs containing the pyrazole nucleus are known in the market. Methods: The 3-phenyl-1H-pyrazol-5(4H)-one was the key starting compound for many heterocyclic reactions to produce substituted and fused pyrazole derivatives. Results: Antiproliferative activities of the produced compounds against six cancer cell lines A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460 were measured through which compounds showed high inhibitions. The most promising compounds were tested against tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR). Structure-Activity Relationship (SAR) was rationalized by looking at the varying structural features of the molecules. In addition, the most active compounds were selected for Pim-1 inhibition. Conclusion: Thirty-nine pyrazole derivatives were synthesized. Nine of them 8b, 9, 12b, 12d, 14b, 15b, 18d, 18f, 19b, and 21d were the most active compounds toward the selected cancer cell lines. Compounds 12b, 14b, 18d, 18f, and 21d showed high inhibitions toward the tyrosine kinases, whereas compounds 14b, 18d, and 18f were the most potent inhibitors of Pim-1.

Stomatin-like Protein 2 Promotes Tumor Cell Survival by Activating the JAK2-STAT3-PIM1 Pathway, Suggesting a Novel Therapy in CRC

Despite intensive efforts, a considerable proportion of colorectal cancer (CRC) patients develop local recurrence and distant metastasis. Stomatin-like protein 2 (SLP-2), a member of the highly conserved stomatin superfamily, is upregulated across cancer types. However, the biological and functional roles of SLP-2 remain elusive in CRC. Here, we report that high SLP-2 expression was found in CRC tissues and was linked to tumor progression and tumor cell differentiation. Additionally, high SLP-2 expression correlated with poor overall survival (OS) in CRC patients (p < 0.001). SLP-2 knockout (SLP-2KO), generated by CRISPR/Cas9, reduced cell growth, migration, and invasion; induced apoptosis in CRC cells; and reduced tumor xenograft growth in vivo. A 181-compound library screening showed that SLP-2KO produced resistance to JAK2 inhibitors (NVP-BSK805 and TG-101348) and a PIM1 inhibitor (SGI-1776), revealing that the JAK2-STAT3-PIM1 oncogenic pathway was potentially controlled by SLP-2 in CRC. In vitro and in vivo, TG-101348 combined with SGI-1776 was synergistic in CRC (combination index [CI] < 1). Overall, our findings suggest that SLP-2 controls the JAK2-STAT3-PIM1 oncogenic pathway, offering a rationale for a novel therapeutic strategy with combined SGI-1776 and TG-101348 in CRC. Additionally, SLP-2 may be a prognostic marker and biomarker for sensitivity to JAK2 and PIM1 inhibitors.

PIM1 inhibitor synergizes the anti-tumor effect of osimertinib via STAT3 dephosphorylation in EGFR-mutant non-small cell lung cancer.

BackgroundAn increasing amount of evidence has demonstrated that combined or multiple targeted therapies could bring about more durable clinical outcomes, and it is known that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance is related to bypass activation. This study aims to explore a specific solution for third-generation EGFR-TKI resistance caused by bypass activation, and to examine the antitumor effects of the combination of a novel inhibitor CX-6258 HCl with osimertinib, along with its underlining mechanisms.MethodsA bioinformatics analysis was performed to detect the relations between the provirus integration site for Moloney murine leukemia virus 1 (PIM1) expression and prognosis of lung cancer. The EGFR-mutated lung cancer cell lines were treated with the combination of CX-6258 HCl and osimertinib to analyze cell proliferation using the Cell Counting Kit-8, colony formation, and in vivo experiments. Cell migration was analyzed using wound healing and Transwell assays. The apoptosis level was detected using Annexin V–propidium iodide flow cytometry. The expression levels of EGFR and STAT3 were determined using Western blot analysis.ResultsHigh expression level of PIM1 was related to the poor prognosis of non-small cell lung cancer (NSCLC). The combined administration of osimertinib and CX-6258 HCl significantly inhibited cell proliferation and migration and effectively induced apoptosis in lung cancer cells. It was more efficient in suppressing EGFR activation and phosphorylation of STAT3 compared with osimertinib treatment alone. Furthermore, it showed a durable efficacy in a xenograft model.ConclusionsThis study showed that PIM1 is a poor prognostic factor for NSCLC. CX-6258 HCl is a potential molecular inhibitor to sensitize the antitumor effects of osimertinib through the inhibiting of the phosphorylation of STAT3 in NSCLC.

A phase Ib study targeting PIM1 and CDK4/6 kinases in metastatic renal cell carcinoma (PICKRCC).

TPS771 Background: Over the last few years there has been a rapid increase in the clinically relevant agents available to treat metastatic renal cell carcinoma (mRCC). We note, however, that the vast majority of new agents available for mRCC do not exploit new clinical targets or pathways. We believe that the identification of new, clinically relevant targets in RCC will propel the field even further forward, expand treatment options, and lead to improved survival for mRCC patients. SGI-1776, a selective PIM1 kinase inhibitor, has previously been shown to induce reduction in tumor size as monotherapy and in combination with sunitinib in mouse pre-clinical studies of RCC. In our tissue microarray studies a subset of ~26% of RCC showed high staining for PIM1 kinase while only 1% of normal adjacent tissue showed similar high staining. Wildtype PIM1 is constitutively active, thus these data suggest that PIM1 activity is increased in a subset of RCC. Abemaciclib is a potent CDK4/6 inhibitor with an IC50 of 2 and 10 nM, respectively. It is also a potent PIM1 inhibitor with an IC50 of 50 nM. We have shown that abemaciclib decreases cell viability and increases apoptosis in RCC cell lines, and does so at greatest effect in combination with sunitinib. We have also shown that abemaciclib induces regression of RCC tumors in a mouse model of RCC, with the most rapid responses observed when abemaciclib is combined with sunitinib. Based on these data we have opened a phase Ib dose escalation study to determine the safety and tolerability of abemaciclib in combination with sunitinib in patients with mRCC. The study includes an expansion cohort at the recommended phase II dose to evaluate for a signal for efficacy. Pattern and intensity of PIM1 staining in tumor tissue will be evaluated as a potential biomarker of response. We are also collecting blood and urine to evaluate additional potential biomarkers of response. Methods: Clinical trial information: NCT03905889 .[Table: see text]

Structural insights for rational design of new PIM-1 kinase inhibitors based on 3,5-disubstituted indole derivatives: An integrative computational approach

Proviral integration Moloney virus (PIM) 1, 2, and 3 kinases are a family of constitutively active serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. Their overexpression in a variety of human hematopoietic malignancies and solid tumors suggest that inhibition of PIM signaling could provide patients with therapeutic benefit. In this study, a series of 3,5-disubstituted indole derivatives have been systematically studied using three-dimensional quantitative structure-activity relationship (3D−QSAR) analysis, molecular docking simulation, and partial least-squares (PLS) analysis methods to explore the influence of the structural characteristics on the inhibitory activity and use them to propose novel bioactive molecules. The comparative molecular field and comparative molecular similarity indices analyses (CoMFA and CoMSIA) models exhibited a good correlation between the predicted and experimental activities with excellent predictive capability and yielded statistically reliable value (CoMFA: Q2 = 0.535, R2 = 0.987, r2pred = 0.909; CoMSIA: Q2 = 0.785, R2 = 0.989, r2pred = 0.969). Based on the CoMFA and CoMSIA models and docking results, ten novel potent PIM-1 inhibitors (N1–N10) have been designed and the molecular models have validated their inhibitory activities. These results provided strong theoretical guidance for the development of novel PIM-1 inhibitors.

PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer.

Resistance to chemotherapy represents a major obstacle to the successful treatment of non-small-cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live-cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intracellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic antitumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

PIM1 (Moloney Murine Leukemia Provirus Integration Site) Inhibition Decreases the Nonhomologous End-Joining DNA Damage Repair Signaling Pathway in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by the narrowing of pulmonary arteries (PAs). It is now established that this phenotype is associated with enhanced PA smooth muscle cells (PASMCs) proliferation and suppressed apoptosis. This phenotype is sustained in part by the activation of several DNA repair pathways allowing PASMCs to survive despite the unfavorable environmental conditions. PIM1 (Moloney murine leukemia provirus integration site) is an oncoprotein upregulated in PAH and involved in many prosurvival pathways, including DNA repair. The objective of this study was to demonstrate the implication of PIM1 in the DNA damage response and the beneficial effect of its inhibition by pharmacological inhibitors in human PAH-PASMCs and in rat PAH models. Approach and Results: We found in vitro that PIM1 inhibition by either SGI-1776, TP-3654, siRNA (silencer RNA) decreased the phosphorylation of its newly identified direct target KU70 (lupus Ku autoantigen protein p70) resulting in the inhibition of double-strand break repair (Comet Assay) by the nonhomologous end-joining as well as reduction of PAH-PASMCs proliferation (Ki67-positive cells) and resistance to apoptosis (Annexin V positive cells) of PAH-PASMCs. In vivo, SGI-1776 and TP-3654 given 3× a week, improved significantly pulmonary hemodynamics (right heart catheterization) and vascular remodeling (Elastica van Gieson) in monocrotaline and Fawn-Hooded rat models of PAH. We demonstrated that PIM1 phosphorylates KU70 and initiates DNA repair signaling in PAH-PASMCs and that PIM1 inhibitors represent a therapeutic option for patients with PAH.

Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) insertions in cancer genomes have been linked to various forms of focal genomic instability and altered expression of neighboring genes. Here we tested the hypothesis that investigation of HPV insertions in a head and neck cancer squamous cell carcinoma (HNSCC) cell line would identify targetable driver genes contributing to oncogenesis of other HNSCC. In the cell line UPCI:SCC090 HPV16 integration amplified the PIM1 serine/threonine kinase gene ~16-fold, thereby increasing transcript and protein levels. We used genetic and pharmacological approaches to inhibit PIM kinases in this and other HNSCC cell lines. Knockdown of PIM1 transcripts by transfected short hairpin RNAs reduced UPCI:SCC090 viability. CRISPR/Cas9-mediated mutagenesis of PIM1 caused cell cycle arrest and apoptosis. Pharmacological inhibition of PIM family kinases decreased growth of UPCI:SCC090 and additional HNSCC cell lines in vitro and a xenograft UPCI:SCC090 model in vivo. Based on established interactions between intracellular signaling pathways and relatively high levels of gene expression in almost all HNSCC, we also evaluated combinations of PIM kinase and epidermal growth factor receptor (EGFR) inhibitors. Dual inhibition of these pathways resulted in supra-additive cell death. These data support clinical testing of PIM inhibitors alone or in combination in HNSCC.

Integrated molecular docking, 3D QSAR and molecular dynamics simulation studies on indole derivatives for designing new Pim-1 inhibitors

Pim-1 is one of the isoforms of pim proteins comprising pim-1, pim-2 and pim-3. It was basically recognized as proviral integration moloney murine leukemia virus which is associated with T-cell lymphomogenesis. Pim-1 is known to play a crucial role in cell cycle progression and acts as downstream target for the JAK/STAT signaling pathway. Recently it has emerged as a hopeful therapeutic target for cancer treatment as deregulation or over expression of pim causes hematologic cancers. In present article molecular docking based three dimensional quantitative structure and activity relationship and molecular dynamics simulation studies have been carried out on indole derivatives reported as pim-1 inhibitors. Initially docking was carried out to obtain the receptor specific orientation of the molecules and later to understand the structural requirements of pim-1 inhibitors robust 3 D QSAR models were built using CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods. The reliability of the models was established from conventional (r2) and cross validated (q2) values of 0.982, 0.524 for CoMFA and 0.974, 0.586 for CoMSIA respectively. Further the predictive ability of the model was evaluated using a test set of 17 molecules. The docking studies revealed that interaction with Glu 121 is vital for binding of inhibitors to pim-1. Based on the outcome of the results new molecules with improved activity were designed. Furthermore, MD simulations were also performed to examine the stability of interactions and investigate the pivotal role of Glu 121.

Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation.

Rationale: Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor and could mediate the activities of multiple transcriptional factors via its non-canonical functions. However, the underlying mechanism of posttranscriptional modification on the non-canonical functions of FBP1 remains elusive.Methods: We employed immunoaffinity purification to identify binding partner(s) and used co-immunoprecipitation to verify their interactions. Kinase reaction was used to confirm PIM2 could phosphorylate FBP1. Overexpression or knockdown proteins were used to assess the role in modulating p65 protein stability. Mechanistic analysis was involved in protein degradation and polyubiquitination assays. Nude mice and PIM2-knockout mice was used to study protein functions in vitro and in vivo.Results: Here, we identified Proviral Insertion in Murine Lymphomas 2 (PIM2) as a new binding partner of FBP1, which could phosphorylate FBP1 on Ser144. Surprisingly, phosphorylated FBP1 Ser144 abrogated its interaction with NF-κB p65, promoting its protein stability through the CHIP-mediated proteasome pathway. Furthermore, phosphorylation of FBP1 on Ser144 increased p65 regulated PD-L1 expression. As a result, phosphorylation of FBP1 on Ser144 promoted breast tumor growth in vitro and in vivo. Moreover, the levels of PIM2 and pSer144-FBP1 proteins were positively correlated with each other in human breast cancer and PIM2 knockout mice.Conclusions: Our findings revealed that phosphorylation noncanonical FBP1 by PIM2 was a novel regulator of NF-κB pathway, and highlights PIM2 inhibitors as breast cancer therapeutics.

Direct Phosphorylation and Stabilization of HIF-1α&nbsp;By PIM1 Kinase Drives Angiogenesis in Solid Tumors

Angiogenesis is essential for sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Thus, understanding mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases (PHDs) to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Overexpression of PIM1 is sufficient to stabilize HIF-1α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner in vivo. CRISPR mutants of HIF-1α (Thr455D) showed increased tumor growth, proliferation and angiogenesis. Moreover, T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

A new hope for neuroblastoma treatment?

Combined ALK and PIM1 inhibition may treat high-risk neuroblastomas.

Inhibition of PIM2 in liver cancer decreases tumor cell proliferation invitro and invivo primarily through the modulation of cell cycle progression.

Liver cancer is the fourth leading cause of cancer-related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2-specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST-1 assays and spheroid assays), 3D-colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT-qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh-7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)-complexed siRNA. The knockdown of PIM2 resulted in potent anti-proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh-7 cells. In vivo, the therapeutic nanoparticle-mediated delivery of PIM2 siRNA led to profound anti-tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.

Abstract B132: Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy

Abstract Oncogene-targeted therapies are initially effective in most patients with BRAF-mutated melanoma. However, patients frequently develop resistance during therapy. At the time of therapy resistance, they are also less likely to respond to immune checkpoint inhibitors (ICI) with response rates ranging from 0-12%, compared to 40-60% when used in the first line. This may be in part be due to rapid tumor growth that outpaces pharmacodynamics of ICI. New drugs that directly control tumor growth and effectively enhance ICI activity are therefore needed. We report that the small molecule CX-6258 has potent activity against both RAF/MEK inhibitor (RMS) and resistant (RMR) BRAF-mutant melanoma cell lines. CX-6258 is annotated as a pan-PIM kinase inhibitor, with an EC50 corrected for growth rate (GR50) of ~200 nM; in contrast, other PIM inhibitors had no in vitro activity, suggesting that PIMs may not be the true target in melanoma - we therefore sought to systematically study the targets of this compound. Using an unbiased kinase profiling approach, we identified Haspin Kinase (HASPIN) as the key target of CX-6258 with an IC50~10nM. Haspin kinase (Haspin) is a poorly understood mitotic kinase, whose only well-defined function is phosphorylation of Histon H3 at Thr3 (pH3T3). Treatment with CX-6258 resulted in reduced proliferation, reduced phosphorylation of pH3T3, frequent formation of micronuclei (MN), recruitment of cGAS and activation of the cGAS-STING-pathway. By using CRISPR/CAS9 knock outs of PIM kinase we show that these targets are not required for CX-6258 function. Furthermore, siRNA mediated knock-down of HASPIN phenocopied CX-6258. In murine models, CX-6258 induces a potent cGAS dependent type-I-interferon response in tumor cells, increases IFNγ-producing CD8+ T-cells, reduces Tregs, and enhances responses to ICIs in vivo. HASPIN is more strongly expressed in malignant compared to healthy tissue and its inhibition by CX-6258 has minimal toxicity in ex vivo expanded human tumor-infiltrating-lymphocytes (TILs) and in vitro differentiated human neurons, suggesting a potential therapeutic index for anti-cancer therapy. CX-6258 also shows activity in several Ewing sarcoma and multiple myeloma cell lines with. In summary, HASPIN inhibition may overcome drug resistance in melanoma, synergize with ICIs and target a vulnerability in different cancer lineages. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Kai Wucherpfennig, Peter K. Sorger, Benjamin Izar. Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B132. doi:10.1158/1535-7163.TARG-19-B132

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

The pan-PIM inhibitor INCB053914 displays potent synergy in combination with ruxolitinib in models of MPN

AbstractAberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration–approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.

FLT3-ITD Activates RSK1 to Enhance Proliferation and Survival of AML Cells by Activating mTORC1 and eIF4B Cooperatively with PIM or PI3K and by Inhibiting Bad and BIM.

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously found that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/AKT pathways to promote proliferation and survival by enhancing the eIF4F complex formation required for cap-dependent translation. Here, we show that, in contrast to BCR/ABL causing Ph-positive leukemias, FLT3-ITD distinctively activates the serine/threonine kinases RSK1/2 through activation of the MEK/ERK pathway and PDK1 to transduce signals required for FLT3-ITD-dependent, but not BCR/ABL-dependent, proliferation and survival of various cells, including MV4-11. Activation of the MEK/ERK pathway by FLT3-ITD and its negative feedback regulation by RSK were mediated by Gab2/SHP2 interaction. RSK1 phosphorylated S6RP on S235/S236, TSC2 on S1798, and eIF4B on S422 and, in cooperation with PIM, on S406, thus activating the mTORC1/S6K/4EBP1 pathway and eIF4B cooperatively with PIM. RSK1 also phosphorylated Bad on S75 and downregulated BIM-EL in cooperation with ERK. Furthermore, inhibition of RSK1 increased sensitivities to BH3 mimetics inhibiting Mcl-1 or Bcl-2 and induced activation of Bax, leading to apoptosis, as well as inhibition of proliferation synergistically with inhibition of PIM or PI3K. Thus, RSK1 represents a promising target, particularly in combination with PIM or PI3K, as well as anti-apoptotic Bcl-2 family members, for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML.