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4138 Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Diseases, Lymphoid Malignancies, Biological Processes, pathogenesis
Monday, December 11, 2023, 6:00 PM-8:00 PM

Kohei Kume, PhD1*, Jaewoong Lee, PhD1,2*, Zhangliang Cheng1*, Mark E Robinson, PhD1*, Etienne Leveille, MD-PhD1*, Kadriye Nehir Cosgun, PhD1*, Lai N Chan, PhD1, Yanzhi Feng1*, David Fonseca Arce1*, Dhruv Khanduja1*, Derek Toomre, PhD3* and Markus Müschen, MD1

1Center of Molecular and Cellular Oncology, Yale University, New Haven, CT
2School of Biosystems and Biomedical Sciences, Korea University, Seoul, Korea, Republic of (South)
3Department of Cell Biology and Biophysics, Yale University, New Haven, CT

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca2+-signals.

Significance: To interrogate Ca2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time (Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt’s lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin’s disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin (Figure A).

Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca2+ oscillations by blue light pulses (Figure B). To model frequency-modulated Ca2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h (Figure B).

Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11L232LI, MYD88L265P and IKK2S177E/S181E not only reduced the frequency of autonomous Ca2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.

Disclosures: No relevant conflicts of interest to declare.

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