Hbxip (Lamtor5) is essential for embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1

Hbxip, also named Lamtor5, has been well characterized as a transcriptional coactivator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5. Using Hbxip knockout embryonic stem cells (ESCs), we showed that depletion of Hbxip compromises the self-renewal of ESCs, with reduced expression of pluripotency genes, reduced cell proliferation, and decreased colony forming capacity. In addition, Hbxip-/- ESCs are defective in differentiation, particularly ectodermal and mesodermal differentiation. Consistently, Hbxip-/- epiblast fails to differentiate properly, indicated by sustained expression of Oct4 in E8.5 Hbxip-/- epiblast. Mechanistically, in ESCs, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, display differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/-, and p18-/- mice, lacking subunits of the Ragulator complex, also share similar phenotypes, embryonic lethality and retarded growth around E7-8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy. We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival. Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment. Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. Disclosures Neel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Inhibition of PINK1-Dependent Mitochondrial Quality Control Pathways Induces Senescence of Acute Myeloid Leukemia Stem Cells

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Novel therapies that target LSCs have the potential to improve clinical outcomes. An important step towards achieving this goal is identifying the fundamental processes that regulate cell fate decisions in LSCs. Perturbation of these processes may impair LSC activity and form the basis of novel therapies. Here, we investigated the role of mitochondrial quality control in the regulation of LSCs by inhibiting PTEN-induced kinase 1 (PINK1). PINK1 is a serine/threonine kinase that serves as a critical sensor of mitochondrial damage and is at the apex of multiple quality control pathways that maintain mitochondrial homeostasis. Damage to mitochondria results in activation of PINK1 on the outer mitochondrial membrane, which in turn triggers mitochondrial fission and mitophagy. We first determined if the overall quality of the mitochondrial pool differs between LSCs and their more differentiated progenies (henceforth termed "non-LSCs") in two human AML cultures known as OCI-AML-8227 and OCI-AML-21. In both cultures, LSCs assayed by xenotransplantation are found only in the CD34 +CD38 -fraction and not in the CD34 +CD38 + and CD34 - fractions. We monitored the turnover rate of the mitochondrial pool in the different fractions by expressing MitoTimer, a mitochondria-targeted fluorescent protein that matures from green to red fluorescence over 48 hours. A high ratio of green to red fluorescence is indicative of active mitochondrial protein turnover and high overall mitochondrial quality. In both cell lines, the fluorescence ratio was highest in the CD34 +CD38 - fraction compared with the other fractions, suggesting that LSCs maintain a higher quality pool of mitochondria than non-LSCs. Based on the above findings, we hypothesized that PINK1-dependent mitochondrial quality control mechanisms are involved in the regulation of LSC fate. To test this hypothesis, we silenced the expression of PINK1 in OCI-AML-8227 cells using lentiviral vectors expressing validated shRNAs under a doxycycline-inducible promoter. Depletion of PINK1 shifted the MitoTimer fluorescence from green to red, reduced oxygen consumption rate, and disrupted mitochondrial ultrastructure in all cell fractions, consistent with a reduction in mitochondrial quality. Unexpectedly, PINK1 downregulation resulted in a block in differentiation and cell cycle arrest at G1/G0 phase in CD34 +CD38 - cells. Re-expression of PINK1 was unable to reverse the cell cycle arrest, suggestive of a state of cellular senescence. Indeed, we observed other hallmarks of senescence including an increase in p21 WAF1 and p16 INK4a expression and SA-β-gal activity. To determine the mechanism by which PINK1 depletion causes senescence, we performed RNA sequencing analysis of sorted CD34 +CD38 - cells expressing PINK1 shRNAs. This analysis revealed a marked decrease in the expression of MYC target genes, with TERT (Telomerase Reverse Transcriptase) being the most downregulated gene. Consistent with the role of TERT in telomere maintenance and telomere shortening as a trigger of senescence, we found that PINK1 knockdown decreased telomere length in CD34 +CD38 - cells, and overexpression of TERT effectively rescued the senescence phenotype. These findings collectively indicate that inhibition of PINK1-dependent mitochondrial quality control pathways induces senescence of LSCs through telomere shortening. To determine whether these changes translated to a reduction in functional LSC activity, we performed colony forming unit assays and serial xenotransplantation assays in immunodeficient NSG mice. Depletion of PINK1reduced the colony forming capacity and engraftment potential of 3 primary AML samples in primary and secondary recipients. Importantly, PINK1 depletion had minimal impact on the colony forming capacity and engraftment potential of normal CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from cord blood, suggestive of a therapeutic window in vivo. In summary, our results demonstrate that mitochondrial quality control pathways regulate cell fate decision in LSCs. Inhibition of PINK1 activity impairs LSC activity by inducing senescence, while sparing normal HSPCs. Our findings provide the basis for exploring PINK1 as a therapeutic target against LSCs in AML. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding.

Modulation of Colorectal Tumor Behavior via lncRNA TP53TG1-Lipidic Nanosystem

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the p53-mediated DNA damage and the intracellular localization of the oncogenic YBX1 protein. However, to translate this finding into the clinic as a gene therapy, it is important to develop effective carriers able to deliver exogenous lncRNAs to the targeted cancer cells. Here, we propose the use of biocompatible sphingomyelin nanosystems comprising DOTAP (DSNs) to carry and deliver a plasmid vector encoding for TP53TG1 (pc(TP53TG1)-DSNs) to a colorectal cancer cell line (HCT-116). DSNs presented a high association capacity and convenient physicochemical properties. In addition, pc(TP53TG1)-DSNs showed anti-tumor activities in vitro, specifically a decrease in the proliferation rate, a diminished colony-forming capacity, and hampered migration and invasiveness of the treated cancer cells. Consequently, the proposed strategy displays a high potential as a therapeutic approach for colorectal cancer.

CAMK2G is identified as a novel therapeutic target for myelofibrosis

Although JAK1/2 inhibition is effective into alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here we established iPS cells derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were utilized for chemical screening and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutations recurrently detected in MF patients were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 gamma subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515Ldecreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

Loss of RANBP3L leads to transformation of renal epithelial cells towards a renal clear cell carcinoma like phenotype

Background Renal cell carcinomas (RCC) are characterized by the deregulation of several hundred hyperosmolality-responsive genes. High expression of a subset of these genes including the Ran binding protein 3 like (RANBP3L) is linked to a favorable prognostic outcome in RCC. However, the cellular function of RANBP3L remains largely unknown. Methods We used CRISPR/Cas9-mediated gene editing to generate functional deletions of the Ranbp3l and nuclear factor of activated T cells 5 (Nfat5) gene loci in a murine renal cell line. The NFAT5-KO cells were used to assess the regulation of Ranbp3l by NFAT5 using immunofluorescence, RNA-Seq and promoter assays. RANBP3L-deficient cells were analyzed for changes in cell morphology, proliferation, migration and colony-forming capacity using immunofluorescence and live cell imaging. RANPB3L-dependent changes in gene expression were identified by RNA-Seq. Results We show that NFAT5 directly regulates Ranpb3l under hyperosmotic conditions by binding its promoter. Functional analysis of RANBP3L-deficient cells revealed a loss of epithelial structure, an increased cell migration behavior and colony forming capacity, accompanied by massive alterations in gene expression, all of which are hallmarks for tumor cells. Strikingly, a RANBP3L dependent signature of 60 genes separated samples with clear cell carcinoma (KIRC) from papillary (KIRP), chromophobe renal carcinoma (KICH) and healthy tissue. Conclusions Loss of RANBP3L induces a tumor like phenotype resembles RCC, especially KIRC, on the morphological and gene expression level and might promote tumor development and progression. Therapeutic reconstitution or elevation of osmoregulated RANBP3L expression might represent a novel treatment strategy for RCC or KIRC.

Characterisation of Regional Human Meniscal Progenitor Cells

Background The surgical treatment of meniscus injury has represented a clinical challenge for decades. Stimulating meniscus regeneration using transplanted meniscal progenitor cells has been suggested as a promising new strategy. However, there is a lack of studies which decisively identify and characterise progenitor cell populations in human meniscus tissues. Methods In this study, donor-matched progenitor cells were isolated via selective fibronectin adhesion from the avascular (PAvas) and vascular (PVas) regions of the meniscus and chondroprogenitors (PChs) from articular cartilage (n=5 donors). In addition, whole mixed populations of cells (MAvas, MVas, MChs) from the same regions were obtained by standard isolation techniques for comparison. The colony formation efficacy of PAvas, PVas and PChs was monitored using Cell-IQ® live cell imaging. Proliferation rates of progenitors were compared with their mixed population counterparts. Cell surface markers indicative of mesenchymal stromal cells (MSCs) profile and progenitor markers were characterised by flow cytometry in all populations. The chondrogenic capacity was assessed via pellet culture assays and measuring chondrogenic gene expression levels, GAG/DNA content and morphology. Results All meniscal progenitor and chondroprogenitor populations showed colony forming capacity in monolayer culture, whereas mixed populations were distributed randomly at passage 0. PVas had significantly lower population doubling times compare to MVas and proliferated faster than PAvas and PChs based on colony forming efficacy. Progenitor populations showed significantly higher positivity for CD49b and CD49c compared to their mixed population counterparts and PChs had a higher positivity level of CD166 compared to mixed chondrocytes. Collagen types II and X expression was significantly downregulated in pellets formed by progenitor populations. GAG/DNA analysis demonstrated that progenitor cells generally produced more GAG than mixed populations. Conclusions Our study demonstrates that the human meniscus contains meniscal progenitor populations in both the avascular and vascular regions. Meniscal progenitors derived from the vascular region exhibit enhanced proliferative and chondrogenic characteristics compared to those from the avascular region; this may associate with the enhanced meniscal healing potential in the vascular region. These findings build on the body of evidence which suggests that meniscal progenitors represent an attractive cell therapy strategy for meniscal regeneration.

Scaffold-mediated CRISPR-Cas9 delivery system for acute myeloid leukemia therapy

Leukemia stem cells (LSCs) sustain the disease and contribute to relapse in acute myeloid leukemia (AML). Therapies that ablate LSCs may increase the chance of eliminating this cancer in patients. To this end, we used a bioreducible lipidoid-encapsulated Cas9/single guide RNA (sgRNA) ribonucleoprotein [lipidoid nanoparticle (LNP)–Cas9 RNP] to target the critical gene interleukin-1 receptor accessory protein (IL1RAP) in human LSCs. To enhance LSC targeting, we loaded LNP-Cas9 RNP and the chemokine CXCL12α onto mesenchymal stem cell membrane–coated nanofibril (MSCM-NF) scaffolds mimicking the bone marrow microenvironment. In vitro, CXCL12α release induced migration of LSCs to the scaffolds, and LNP-Cas9 RNP induced efficient gene editing. IL1RAP knockout reduced LSC colony-forming capacity and leukemic burden. Scaffold-based delivery increased the retention time of LNP-Cas9 in the bone marrow cavity. Overall, sustained local delivery of Cas9/IL1RAP sgRNA via CXCL12α-loaded LNP/MSCM-NF scaffolds provides an effective strategy for attenuating LSC growth to improve AML therapy.

Jiyuan Oridonin A Overcomes Differentiation Blockade in Acute Myeloid Leukemia Cells With MLL Rearrangements via Multiple Signaling Pathways

Differentiation therapy with all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), has been extremely successful in inducing clinical remission in APL patients. However, the differentiation therapy of ATRA-based treatment has not been effective in other subtypes of AML. In this study, we evaluated a small molecule of ent-kaurene diterpenoid, Jiyuan oridonin A (JOA), on the differentiation blockade in AML cells with the mixed lineage leukemia (MLL) gene rearrangements (MLLr) in MV4-11, MOLM-13 and THP-1 cells. We found that JOA could significantly inhibit the proliferation of MOLM-13, MV4-11 and THP-1 cells. Moreover, JOA promoted cell differentiation coupled with cell-cycle exit at G0/G1 and inhibited the colony- forming capacity of these cells. We showed that the anti-proliferative effect of JOA attributed to cell differentiation is most likely through the martens tretinoin response up pathway in the MOLM-13 cell line, and the hematopoietic cell lineage pathway by the inhibition of c-KIT expression and cell adhesion pathway in the THP-1 cell line. Our findings suggest that JOA could be a novel therapeutic agent against human MLLr acute myeloid leukemia.

LIGHT/LTβR signaling regulates self-renewal and differentiation of hematopoietic and leukemia stem cells

The production of blood cells during steady-state and increased demand depends on the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation. Similarly, the balance between self-renewal and differentiation of leukemia stem cells (LSCs) is crucial in the pathogenesis of leukemia. Here, we document that the TNF receptor superfamily member lymphotoxin-β receptor (LTβR) and its ligand LIGHT regulate quiescence and self-renewal of murine and human HSCs and LSCs. Cell-autonomous LIGHT/LTβR signaling on HSCs reduces cell cycling, promotes symmetric cell division and prevents primitive HSCs from exhaustion in serial re-transplantation experiments and genotoxic stress. LTβR deficiency reduces the numbers of LSCs and prolongs survival in a murine chronic myeloid leukemia (CML) model. Similarly, LIGHT/LTβR signaling in human G-CSF mobilized HSCs and human LSCs results in increased colony forming capacity in vitro. Thus, our results define LIGHT/LTβR signaling as an important pathway in the regulation of the self-renewal of HSCs and LSCs.