Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti-programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti-PD-1 Ab was examined in a P-glycoprotein-knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti-PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti-PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti-PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations.

Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

Efficacy of tyrosine kinase inhibitors on a mouse chronic myeloid leukemia model and chronic myeloid leukemia stem cells.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by constitutively active BCR-ABL1 tyrosine kinase resulting from the t(9;22) Philadelphia translocation. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), is a revolutionary molecular target inhibitor for CML. However, leukemic stem cells (LSCs) eventually become resistant to imatinib and thereby cause relapse. The next-generation BCR-ABL1 TKI dasatinib is also unable to eliminate CML LSCs. On the other hand, the third-generation BCR-ABL1 TKI ponatinib is not well studied in terms of its efficacy on CML LSCs. Here, we evaluate the efficacy of ponatinib against CML LSC-containing lin-Sca-1+c-Kit+ (LSK) cells using a mouse CML-like model. To this end, we compared the efficacy of imatinib, dasatinib, and ponatinib on CML LSK cells and showed that ponatinib is more effective at eliminating CML LSK cells. Our results suggest that ponatinib could be potentially useful for achieving treatment-free remission in CML patients.