The Dual PI3K-δ/γ Inhibitor Duvelisib in Combination with the Bcl-2 Inhibitor Venetoclax Shows Promising Responses in Richter Syndrome-PDX Models
Authors: Iannello Andrea , Arruga Francesca , Vitale Nicoletta , Marchi Elisa , Furman Richard R. , Allan John N. , Coma Silvia , Pachter Jonathan A. , Deaglio Silvia , Vaisitti Tiziana 
Published: November 2019

Richtersyndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, typically a diffuse large B cell lymphoma (DLBCL) with poor prognosis and without effective therapies. Chemotherapy, as well as novel inhibitory agents, show only partial and temporary responses, highlighting the need for alternative therapies that can overcome drug resistance. Duvelisib (DUV) is an oral phosphatidylinositol 3-kinase (PI3K)-δ and γ dual inhibitor showing efficacy in patients with refractory CLL. Venetoclax (VEN) is a Bcl-2 selective inhibitor, able to induce apoptosis in CLL cells. The lack of RS cell lines and murine models has limited understanding of the molecular mechanisms contributing to the pathogenesis of this disease, impacting also on the development of effective therapies. In our lab, 4 different RS patient-derived xenografts (PDXs) were established, representing useful tools to study the biology of the disease and to test novel therapeutic strategies.
This work was undertaken to investigate expression and activity of the PI3K pathway and Bcl-2, and to test the ex-vivo and in vivo efficacy of DUV and VEN, alone or in combination, in our RS-PDX models.
The 4 RS-PDX models are characterized by heterogeneous PI3K expression. RS1316 and IP867 expressed high levels of both p110δ and p110γ catalytic subunits. In contrast, RS9737 expressed low levels of both the PI3K catalytic subunits, while RS1050 expressed the p110δ subunit alone, but lacked p110γ and Bcl-2.
We started by assessing the ex-vivo efficacy of DUV and VEN on RS-PDX-derived cells. Cells from the 4 RS-PDX models were obtained from disrupted tumor masses and immediately treated with DUV (5µM), VEN (25nM) or their combination. The DUV/VEN combination induced the highest levels of apoptosis in RS1316, RS9737 and IP867/17 cells at 24 and 48 h after treatment. Western blot confirmed the downregulation of Bcl-2 and Mcl-1, along with Caspase-3 and PARP-1 cleavage. In contrast, no effect was observed on RS1050 cells, which lack p110γ and Bcl-2, making this model intrinsically resistant to DUV and VEN treatment.
Next, the effects of PI3K-δ/γ and Bcl-2 inhibition were examined in vivo on the RS1316-PDX model, selected as the best ex-vivo responder. NOD/SCID/gamma chain-/-(NSG) mice were subcutaneously injected with RS1316 cells. When tumor masses were palpable, animals were treated daily for 7 days including 2 holidays treatment with DUV (100 mg/kg p.o.), VEN (50 mg/kg p.o.) or their combination. In mice that received the combination, tumors were of smallest volume (4 out of 6 tumors were completely absent) and had the least viable cells, as highlighted by increment of cleaved Caspase-3 immunohistochemistry signal. In a second set of experiments, tumor masses were left to grow after 10 consecutive treatments and monitored daily. Used as single agents, DUV and VEN significantly reduced tumor growth, prolonging survival for 5 days compared to controls (mean survival 42 days in the control condition vs 47 in the single treatments). The combination of the two drugs was even more efficacious, leading to a complete remission in all the mice during treatment (Fig. 1). Tumor masses eventually grew back after cessation of treatment, but the combination significantly prolonged survival compared to vehicle or single-treatment groups (mean survival 56 days, p<0.05). Interestingly, RS1316 cells isolated from treated animals after regrowth continued to respond to ex-vivo treatmentwith the same compounds, suggesting that longer treatment may be needed to achieve more durable responses.
In conclusion, DUV and VEN treatment combination, simultaneously inhibiting PI3K-δ/γ and Bcl-2, induced RS tumor regression, offering a promising treatment combination, at least for RS patients expressing high levels of PI3K gamma and delta subunits and Bcl-2. These results pave the way for clinical testing of duvelisib and venetoclax combination for patients with RS.
Figure 1

Disclosures
Furman: AstraZeneca: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Acerta Pharma: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Beigene: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy. Allan:Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Janssen: Consultancy, Honoraria; Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coma:Verastem Inc: Employment. Pachter:Verastem Inc: Employment. Deaglio:VelosBio Inc.: Research Funding; iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding. Vaisitti:Verastem Inc: Research Funding; VelosBio Inc.: Research Funding.

OffLabel Disclosure:
Duvelisib: PI3K gamma and delta inhibitor Venetoclax: bcl2 inhibitor