Dimitrios Tsallos 2021

Counteracting chemotherapy-induced thrombocytopenia

Dimitrios Tsallos

Institute for Molecular Medicine Finland FIMM, University of Helsinki

SUMMARY
Approximately two-thirds of patients with hematologic cancers and one-fourth of patients
with solid tumors experience treatment-related thrombocytopenia with implications in
patient mortality and morbidity. To protect thrombocytes during cancer treatment, we
explored the combination of common cytotoxic treatments with eltrombopag (EP); an
approved treatment for immune thrombocytopenia. To study the drug response of early
thrombocytes, megakaryocytes, we developed a flow cytometry screening method that
utilizes expanded CD34+ cells from healthy donors. We observed the most beneficial effect
from the combination of EP with pan-BCL-2 inhibitor navitoclax. Other common cancer
treatments, including platin based treatments, topoisomerase inhibitors and nucleoside
analogs showed a moderate benefit, while azacytidine and ruxolitinib had strong
counteraction. Finally, we established the molecular mechanisms behind azacytidine/EP
counteraction, and how navitoclax/EP could benefit patients.
AIMS OF THE STUDY
1. To develop a quantitative method to study chemotherapy-induced myelosuppression (1).
2. To describe the effect on MKs for common anti-neoplastic treatments combined with EP.
3. To evaluate the mechanism of action of characteristic treatment combinations with EP,
suggesting an effective treatment option for future clinical trials.

METHODS
1. Primary samples from our systems medicine study of leukemia have been collected
analysed and stored from bone marrow aspirates from more than 150 patients together
with clinical information. Exome and RNA sequencing together with ex vivo drug sensitivity
profiling against more than 300 compounds is available for investigation.
2. Fresh cord blood samples were used according to the needs for MK drug screening method
developed by us which is described briefly in the results section6.
3. Using the iQue screener Plus (Sartorius) we are able to conduct high-throughput flow
cytometry experiments to evaluate sensitivity to treatment for selected populations. We
use various gating strategies and multi-colour antibody panels to identify maturing
megakaryocytes and myeloid progenitors. Similarly, we studied the signalling patterns of
selected populations we designed multi-colour flow cytometry antibody panels that stain
both intracellular and extracellular markers.
4. A drug sensitivity score (DSS) was calculated based on a modified area under the doseresponse
curve calculation as described earlier. R package was used for the calculation of
IC50, DSS, statistical significance testing and plotting results.

RESULTS
Eltrombopag Promotes Megakaryocyte Survival and Signaling in the Presence of Specific
Cytotoxic Agents
To study the effect of eltrombopag (EP) treatment on megakaryocytes (MK) in presence of
cytotoxic agents, we screened compounds in 5 concentrations with 10-fold increase alone or
in combination 1.8 μM EP. For this screening process we developed a high-throughput flow
cytometry method that utilizes expanded CD34+ cells from healthy donors (1, aim 1). The
addition of EP to the treatments allows MKs to overcome the cytotoxic effects of the tested
agents and proliferate in their presence. The results from drug screening assay matched with
signaling activation analysis suggest three distinct treatment groups relating to response in
combination with EP. The most promising treatments allowed for complete signal activation
by EP were of apoptotic modulators. Common treatments mildly inhibited EP signaling
activation to resemble TPO activation, that shows reduced AKT and ERK activation on mature
cells. Coupled with viability results platinum-based treatments, topoisomerase inhibitors and
nucleoside analog gemcitabine suggest a medium response group with potential to benefit
patients when combined with EP. Finally, JAK inhibitor ruxolitinib, the hypomethylating agent
azacitidine and paclitaxel inhibit proliferation triggered by EP (submitted manuscript aim 2).
We further investigated the possibility to combine EP with navitoclax; a promising treatment
that was halted in development due to severe thrombocytopenia. The combination shown
promise in healthy cells and seems effective in various malignancies. We examined the
further use of EP in leukemic cells and identified that signal activation of TPO receptor leads
to alternative splicing of BCL2L1 and increases the expression of Bcl-xS which inhibits the
longer transcript and target of navitoclax Bcl-xL. The parallel stimulation of growth of healthy
cells and the potentiation of navitoclax effect on leukemic cells and makes this a great
treatment combination (finalizing experiments aim 3).
Azacitidine mediated activation of innate immunity impairs megakaryopoiesis
We found that azacitidine triggers a rapid and transient reduction of RNA methylation,
concomitant accumulation of double-stranded RNA species, activated the immune pathogen
sensor TLR3 and increased IFNα/β signaling. Megakaryocytic lineage-primed progenitor cells
expressing high levels of IFN-I receptors elicited STAT1/SOCS1-dependant downstream
signaling, which impaired thrombopoietin receptor (TPO-R) stimulation and megakaryocytic
progenitor cell growth and differentiation. Our findings directly implicate viral mimicry as a
root cause for AZA-mediated thrombocytopenia (submitted manuscript aim 3).
Graduation time is expected during the summer of 2022.

REFERENCES
1. Javarappa, K. K.; Tsallos, D.; Heckman, C. A. A Multiplexed Screening Assay to Evaluate
Chemotherapy-Induced Myelosuppression Using Healthy Peripheral Blood and Bone Marrow. SLAS
Discov 2018, 23, 687–696.
2. Vlachodimitropoulou, E.; Chen, Y.-L.; Garbowski, M.; et al. Eltrombopag: a Powerful Chelator of
Cellular or Extracellular Iron(III) Alone or Combined with a Second Chelator. Blood 2017, 130, 1923–
1933.
3. Xavier-Ferrucio, J.; Scanlon, V.; Li, X.; et al. Low Iron Promotes Megakaryocytic Commitment of
Megakaryocytic-Erythroid Progenitors in Humans and Mice. Blood 2019, 134, 1547–1557.