Myeloma treatment with IMiDs is associated with enhanced platelet reactivity 13th March 2019 Dr Dalia Khan

Introduction Multiple myeloma (MM) associated with elevated levels of venous and arterial thromboses Treatment with immunomodulatory drugs (IMiDs) further increases thrombosis risk Anti-platelet agents offer more effective thromboprophylaxis in patients with IMiDs compared with other indications Suggests platelets are of central importance in IMiD associated thrombosis Venous and arterial thrombosis are both more prevalent in patients with myeloma, with hazard ratios of 7.5 and 1.9, respectively, compared to healthy controls. Thrombosis is correlated with reduced overall survival, and considerable morbidity including neurological compromise with stroke, post thrombotic syndrome, and pulmonary hypertension.(3–5) The 5% pa incidence of spontaneous thrombosis in patients with myeloma is elevated to as much as 58% pa by immunomodulatory drugs (IMiDs; e.g. Lenalidomide, Pomalidomide, Thalidomide) in combination with steroids.(6) As a result, thrombo-prophylaxis with aspirin or low molecular weight heparin (LMWH) is now standard of care when IMiD therapy is initiated. Despite this, many patients who receive thromboprophylaxis with Aspirin or LMWH experience ‘breakthrough’ VTEs. The phase 3 Myeloma XI trial reported that VTE rates were 12% with both Thalidomide and Lenalidomide therapy, despite 88% of patients receiving thromboprophylaxis. These data indicate that current anti-coagulation strategies do not effectively target the underlying cause of thrombosis in these patients. Although LMWH offers the greatest documented reduction in IMiD-associated thrombosis risk in myeloma, the efficacy of aspirin is much greater than that seen in idiopathic VTE. (1)In keeping with this, small studies have shown increased markers of platelet activation after exposure to thalidomide. These findings suggest that improved understanding of platelet activation following IMiD exposure may help to direct the development of novel thrombo-prophylaxis strategies (including the development of IMiDs with fewer off-target effects), and this will be the focus of investigation for this project.

Rationale Understanding mechanisms underlying the increased risk would allow: Alteration of anti-platelet/anti-coagulant prophylaxis Refinement of future drug development removing ‘off-target’ effects of IMiDs

Measures of platelet reactivity: Platelet activation/degranulation/ aggregation Courtesy: http://what-when-how.com/acp-medicine/hemostasis-and-its-regulation-part-1/

CRP TRAP-6 U46619 Modified from Rivera et al. Platelet receptors and signalling in the dynamics of thrombus formation Haematologica May 2009, 94 (5) 700-711; DOI: 10.3324/haematol.2008.003178

Assays- Platelet aggregation- traditional From Chan et al. (2018) 96-well plate-based aggregometry, Platelets, DOI: 10.1080/09537104.2018.1445838 and practical haemostasis

Plate based aggregometry From Chan et al. (2018) 96-well plate-based aggregometry, Platelets, DOI: 10.1080/09537104.2018.1445838

Platelet flow cytometry- P selectin exposure indication of alpha granule release Fibrinogen binding upregulation in affinity for integrin αIIbβ3 Annexin V binding: surrogate for Phosphatidylserine exposure site of prothrombinase complex and activation of coagulation cascade Courtesy : John Francis FIHR

Methods: Study design- Population cohorts MGUS cohort Smouldering Multiple Myeloma (SMM) MM cohort: proteasome inhibitor (PI) MM cohort: IMiD + PI + DOAC MM cohort: IMiD + PI + ASA A Baseline Testing: all groups B MM cohorts on treatment 2 weeks after commencing chemotherapy C 4 weeks after commencing chemotherapy D MM cohorts >2 months post commencing chemotherapy

Results No significant difference in baseline measures of platelet reactivity between SMM, new MM and relapsed MM *

IMiD treatment associated with increased Annexin binding IMiD treated patients Non-IMiD treated patients p=0.0007 One way matched ANOVA p=0.0046 One-way matched ANOVA Schedule, groups, means, thinking, speculate, new imid samples etc. Conclude story, further work Baseline 2 weeks 4 weeks Baseline 2 weeks 4 weeks

IMiD Treatment increases fibrinogen binding IMiD treated patients ADP IMiD treated patients CRP CRP P=0.04 P=0.03 Non- IMiD treated ADP Non- IMiD treated CRP No difference with TRAP, numerous Epi non-responders P=0.0006 Two way matched ANOVA: Tukey’s multiple comparison’s test

IMiDs enhance P-selectin exposure IMiD treated patients CRP IMiD treated patients ADP IMiD treated patients ADP P=<0.0001 P=0.0002 P=0.03 P=0.008 Non- IMiD treated patients CRP Non- IMiD treated patients ADP P=0.04 P=0.05 P=0.008 Two way matched ANOVA: Tukey’s multiple comparison’s test

Platelet aggregation not enhanced by IMiDs in vivo IMiD treated Non- IMiD treated

Donor platelet aggregation increased with in vitro addition of IMiDs- 20 minute incubation T vs. 1UM: p=0.02 T vs. 10uM:p=0.0008 T vs. 1UM:p=0.0002 Tvs. 10uM:p<0.0001 T vs. 1UM:p=0.02 T vs. 10uM:p=0.04 Two way matched ANOVA: Tukey’s multiple comparison’s test

In vitro effect of dexamethasone P=<0.0001 P=0.007 P=0.0001 P=0.003 P=0.002 P=0.03

Conclusion Still early matched data for MGUS n=3, MM on IMiDs= 9, MM not on IMiDs=3, SMM n=4. No differences in baseline measures of platelet reactivity between plasma cell dyscrasia groups IMiDs lead to persistent increase in measures of platelet reactivity after 2 weeks of treatment In vitro: effect of IMiDs on platelets occurs within 20 minutes : cell signalling pathways vs. transcription Dexamethasone also leads to increase in platelet reactivity

Future direction Mechanistic studies: ?IMiDs acting on platelet cereblon/other ubiquitin ligases How do IMiDs affect megakaryocyte maturation, migration and ultimate phenotype of platelets produced Do the new CelMods have the same effect on platelet reactivity