Disrupting IL10 Signaling Overcomes Compound Copy Number Variation-Driven Clinical Resistance to CDK4/6 and BTK Inhibition in Mantle Cell Lymphoma

Drug resistance remains a formidable challenge in MCL, largely due to unrestrained proliferation of MCL cells driven by aberrant Cyclin D1 and CDK4 expression. In preclinical studies, inhibition of CDK4/6 not only induces early G1 cell cycle arrest but also reprograms MCL cells for therapy vulnerability, suggesting that CDK4/6 inhibition may deepen and prolong the clinical response to BTK inhibition. We tested this hypothesis in a phase I clinical trial for recurrent MCL and defined the resistant mechanism by integrated longitudinal genomic analysis of individual patients.

Palbociclib (CDK4/6i) was administered on days 1-21 of a 28-day cycle; ibrutinib (BTKi) was given continuously. For longitudinal analysis, sequential tissue and blood specimens from 27 evaluable patients were collected before and during therapy, and on progression. Single cell RNA-seq (scRNA-seq) analysis of PBMCs or the monocytic fractions from bone marrow and lymph node (53 samples) was performed using a MCL-specific in-house reference library. The data were then integrated with whole transcriptome (WTS) and whole exome sequencing (WES) of MCL cells isolated from the same specimens, flowcytometry, immunoblotting, IHC, and CBC /differential to deduce the number of total MCL and immune cells in each specimen.

CDK4/6 Inhibition appears to deepen and prolong the BTKi response, with a CR rate of 42% and 5 patients (2 CR and 3 PR) remained on therapy for ~ 10 years. Longitudinal scRNA-seq (210,000 cells) revealed that MCL cells comprise 4 major transcriptomically distinct clusters with cluster 1 (C1) resembling quiescent normal B cells. RNA velocity analysis further indicated that both C2 and C3 are in late G1, having evaded CDK4/6 inhibition; but only C2 progresses to the proliferating C4. C3 is terminally arrested in late G1 and long-lived with elevated BCL2 and IRF4 expression.

In durable CR, all MCL clusters were depleted as expected. Primary resistance and progression on therapy correlated with a marked expansion of C2 or C3 MCL cells. Integrated longitudinal scRNA-seq analysis with WES and WTS revealed that C3 resistance was due to RB1^+/del CDKN2A^+/del (hemizygous loss of RB1 and CDKN2A), whereas C2 resistance was driven by CDK4^amp-RB1^+/del-CDKN2A^+/del_. Thus, compound copy number variation (cCNV) drives differential resistance to CDK4/6i and BTKi, reinforcing the critical importance of controlling the CDK4/6-RB-CDKN2A axis in MCL therapy.

Durable CR was also associated with maintenance of CD8⁺ T cells and CD8⁺ T effector memory (TEM) cells. Conversely, progression on therapy was accompanied by rapid depletion of CD8⁺TEMs and proliferating (Ki67⁺) CD8⁺ T cell expressing all exhausting markers including LAG3,TIGIT and TIM3. Collectively, our data suggest that CD8⁺ T cell surveillance cooperates with MCL cell intrinsic cCNV to discriminate durable clinical response from resistance to targeting CDK4/6 and BTK.

Moreover, IL10 was produced in bone marrow MCL cells before therapy regardless of the subsequent clinical response, but downregulated in early G1-arrested in vivo in a responding patient. Plasma IL10 was selectively elevated in MCL patients on progression from CR, along with sustained IL10 synthesis in myeloid cells. These novel findings suggest that IL10 promotes resistance to CDK4/6i and BTKi and a new strategy to overcome resistance. Indeed, exogenous IL10 completely abolished the killing of MCL cells from a PR patient by CDK4/6i+ BTKi and CDK4/6i+ PI3Ki ex vivo. IL10 also abrogated cCNV-guided killing of MCL cells C3 - and C2-resistant patients by BTKi+BCL2i, and C3-resistant patients by CDK2i+BCL2i ex vivo. All therapies were restored by disrupting IL10 signaling.

In summary, by integrated longitudinal scRNA-seq analysis of a hypothesis-driven clinical trial, we have provided the first evidence in humans that 1) CDK4/6 inhibition deepens and prolongs the clinical response to BTKi; 2) cCNV of CDK4-RB1-CDKN2A drives differential expansion of resistant C2 MCL cells in transit to proliferating C4 MCL cells or long-lived non-proliferating C3 MCL cells; 3) CD8⁺ T cell maintenance and surveillance cooperates with MCL cell intrinsic cCNV to discriminate durable response from resistance; and 4) IL10 is a key mediator of MCL-Immune cell interaction that promotes resistance. These discoveries have profound implications for genome-guided strategy to overcome drug resistance in MCL.

Inghirami:Daiichi Sankyo: Consultancy. Lee:Kite Pharma: Consultancy. Maddocks:Genentech: Consultancy; AstraZeneca: Consultancy; Genmab: Consultancy; BMS: Consultancy; Gilead/KITE: Consultancy; Lilly: Consultancy; Incyte: Consultancy; Janssen: Consultancy; ADC Therapeutics: Consultancy; AbbVie: Consultancy. Blum:Seattle Genetics: Research Funding; BMS: Research Funding. Ruan:Genentech: Research Funding; BMS: Honoraria, Research Funding; Janssen: Honoraria; AstraZeneca: Honoraria, Research Funding. Leonard:AbbVie, AstraZeneca, Astellas, Bayer, BeiGene,BMS, Calithera, Constellation, Eisai, Epizyme,GenMab, Grail, Incyte, Janssen, Karyopharm, Lilly,Merck, Mustang Bio, Pfi zer, Roche/Genentech,Seagen, Second Genome, Sutro: Consultancy. Bartlett:Washington University School of Medicine: Current Employment; ADC Therapeutics: Research Funding; Autolus: Research Funding; BMS: Research Funding; Celegne: Research Funding; Forty Seven: Research Funding; Gilead: Research Funding; Kite Pharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Foresight Diagnostics: Membership on an entity's Board of Directors or advisory committees. Martin:AbbVie, AstraZeneca, Beigene, Daiichi Sankyo, Genentech, Janssen, Merck, Pepromene: Consultancy.

Palbociclib is a CDK4/6 inhibitor FDA-approved for breast cancer treatment. It was used off-label in combination with ibrutinib in a phase I clinical trial in patients with relapsed/refractory mantle cell lymphoma.