PI3 kinase / mTOR (vistusertib; AZD2014) combinations with palbociclib in TNBC

CDK2high and CDK2low cell subpopulation arise from single cell

Cell cycle lengths (hours) according to CDK2 status (CDK2high vs. CDK2low)

Taselisib and GDC-0077 have increased potency in PIK3CA-mutant cancer cells

Activity of GDC-0077 in combination with Palbociclib and/ or Fulvestrant

Taselisib-mediated degradation of mutant p110a occurs preferentially at the plasma membrane.

Patients with chronic lymphocytic leukemia (CLL) respond well to initial treatment with the Bcell lymphoma 2 (BCL2) inhibitor venetoclax. Upon relapse, they often retain sensitivity to BCL2 targeting, but durability of response remains a concern. We hypothesize that targeting both BCL2 and B-cell lymphoma-extra large (BCLXL) will be a successful strategy to treat CLL, including for patients who relapse on venetoclax. To test this hypothesis, we conducted a pre-clinical investigation of LP-118, a highly potent inhibitor of BCL2 with moderate BCLXL inhibition to minimize platelet toxicity. This study demonstrated that LP-118 induces efficient BAK activation, cytochrome C release, and apoptosis in both venetoclax naïve and resistant CLL cells. Significantly, LP-118 is effective in cell lines expressing the BCL2 G101V mutation and in cells expressing BCLXL but lacking BCL2 dependence. Using an immunocompetent mouse model, Eμ-TCL1, LP-118 demonstrates low platelet toxicity, which hampered earlier BCLXL inhibitors. Finally, LP-118 in the RS4;11 and OSU-CLL xenograft models results in decreases in tumor burden and survival advantage, respectively. These results provide a mechanistic rationale for the evaluation of LP-118 for the treatment of venetoclax responsive and relapsed CLL.

Taselisib depletes mutant p110a protein through ubiquitin and proteasome mechanism in a dose and time dependent manner

RTK activity plays a key role in regulating p110a degradation

Taselisib and GDC-0077 induce mutant p110a and not WT p110a degradation

Bruton's tyrosine kinase (BTK) inhibitors are effective for the treatment of chronic lymphocytic leukemia (CLL) due to BTK's role in B cell survival and proliferation. Treatment resistance is most commonly caused by the emergence of the hallmark BTKC481S mutation that inhibits drug binding. In this study, we aimed to investigate whether the presence of additional CLL driver mutations in cancer subclones harboring a BTKC481S mutation accelerates subclone expansion. In addition, we sought to determine whether BTK-mutated subclones exhibit distinct transcriptomic behavior when compared to other cancer subclones. To achieve these goals, we employ our recently published method (Qiao et al. 2024) that combines bulk DNA sequencing and single-cell RNA sequencing (scRNA-seq) data to genotype individual cells for the presence or absence of subclone-defining mutations. While the most common approach for scRNA-seq includes short-read sequencing, transcript coverage is limited due to the vast majority of the reads being concentrated at the priming end of the transcript. Here, we utilized MAS-seq, a long-read scRNAseq technology, to substantially increase transcript coverage across the entire length of the transcripts and expand the set of informative mutations to link cells to cancer subclones in six CLL patients who acquired BTKC481S mutations during BTK inhibitor treatment. We found that BTK-mutated subclones often acquire additional mutations in CLL driver genes, leading to faster subclone proliferation. When examining subclone-specific gene expression, we found that in one patient, BTK-mutated subclones are transcriptionally distinct from the rest of the malignant B cell population with an overexpression of CLL-relevant genes.

Angioimmunoblastic T-cell lymphoma (AITL), the most common form of peripheral T-cell lymphoma, originates from follicular helper T (Tfh) cells and is notably resistant to current treatments. The disease progression and maintenance, at least in early stages, are driven by a complex interplay between neoplastic Tfh and clusters of B-cells within the tumor microenvironment, mirroring the functional crosstalk observed inside germinal centers. This interaction is further complicated by recurrent mutations, such as TET2 and DNMT3A, which are present in both Tfh cells and B-cells. These findings suggest that the symbiotic relationship between these 2 cell types could represent a therapeutic vulnerability. This review examines the key components and signaling mechanisms involved in the synapses between B-cells and Tfh cells, emphasizing their significant role in the pathobiology of AITL and potential as therapeutic targets.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults. Available treatments have not markedly improved patient survival in the last twenty years. However, genomic investigations have showed that the PI3K pathway is frequently altered in this glioma, making it a potential therapeutic target.Paxalisib is a brain penetrant PI3K/mTOR inhibitor (mouse Kp,uu 0.31) specifically developed for the treatment of GBM. We characterised the preclinical pharmacokinetics and efficacy of paxalisib and predicted its pharmacokinetics and efficacious dose in humans.Plasma protein binding of paxalisib was low, with the fraction unbound ranging from 0.25 to 0.43 across species. The hepatic clearance of paxalisib was predicted to be low in mice, rats, dogs and humans, and high in monkeys, from hepatocytes incubations. The plasma clearance was low in mice, moderate in rats and high in dogs and monkeys. Oral bioavailability ranged from 6% in monkeys to 76% in rats.The parameters estimated from the pharmacokinetic/pharmacodynamic modelling of the efficacy in the subcutaneous U87 xenograft model combined with the human pharmacokinetics profile predicted by PBPK modelling suggested that a dose of 56 mg may be efficacious in humans. Paxalisib is currently tested in Phase III clinical trials.

T-cell prolymphocytic leukemia (T-PLL) is a rare and deadly mature leukemia with few treatment options and adverse prognosis. Currently there is no effective treatment therapy for patients with relapse disease, with an overall survival of less than 6 months. Our group demonstrated a strong T-PLL dependency on the BCL2 family of antiapoptotic proteins using BH3 profiling (2023, ASH Annual Meeting, #4192). We generated an in vivo patient derived T-PLL xenograft (PDX) model by engrafting a relapsed/refractory T-PLL patient sample into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. This model reproduces key human clinicopathological features of T-PLL. Mice develop enlarged spleens, immunohistochemical and flow cytometry studies show engrafted T-PLL tumor cells are CD3+, CD4+ and can be detected in liver, spleen, and peripheral blood (PB). Tumor cells have TCL1 chromosomal rearrangements detected by FISH. The model was expanded and utilized in a pre-clinical trial to evaluate a novel therapeutic combination using Fadraciclib (a CDK2/9 inhibitor) and Venetoclax (BCL2 inhibitor). In vitro evidence from our team has demonstrated an additive/synergistic effect of Venetoclax in combination with Fadraciclib to cause mitochondrial dysfunction and cell death of T-PLL. These drugs inhibit separate, but partially overlapping signaling pathways covering BCL2, and MCL1. In our first set of experiments, we evaluated the effect of Venetoclax and Fadraciclib independently and in combination on the overall survival of T-PLL PDX mice. Mice were enrolled for treatment once human/Total CD45 positive cells reached 5% in PB. Fadraciclib (10mg/kg) and Venetoclax (25mg/kg) administered via gavage once daily for 5 days, 2 days of rest, and repeat for a total of 2 weeks was well tolerated. We demonstrate that therapy with Fadraciclib plus Venetoclax significantly prolonged survival compared with vehicle (p=0.0267, Log-rank (Mantel-Cox) test), and Fadraciclib alone (p=0.0103). We also evaluated the effects of these drugs on the percentage of circulating T-PLL cells in peripheral blood (PB; hCD45+). The Venetoclax and Fadraciclib combination maintained a low percentage of circulating T-PLL cells in PB, (maximum average 4.2%), Fadraciclib (20.8%), Venetoclax (17.9%), and Vehicle (42.2%) during treatment. In conclusion, we have developed a T-PLL PDX model that can be used to reproducibly evaluate the effect of new drugs and combinations for T-PLL in a preclinical setting. Combination therapy with the CDK9 inhibitor Fadraciclib and the BCL2 inhibitor Venetoclax was well tolerated, controlled the burden of circulating T-PLL cells in PB better than independent drugs and vehicle, and improved survival of T-PLL mice that were treated with the combination. Such data can provide the rationale for a clinical trial exploring this combination in patients with relapsed or refractory T-PLL.

Treatments for T-cell prolymphocytic leukemia (T-PLL), while transiently effective in the frontline, have failed to achieve any substantial improvement in the outcomes of this ultra-rare chronic leukemia. Patients with relapsed T-PLL have a dismal prognosis, with a median overall survival (OS) of less than 6 months. For this study we used samples collected from: 8 patients with untreated T-PLL, 2 with relapsed/refractory (R/R) T-PLL, and 1 from a PDX model of R/R T-PLL. Whole-exome sequencing (WES) was used to elucidate the mutational landscape and single cell T-cell receptor sequencing (scTCRseq), coupled with single cell RNA sequencing (scRNA-seq), was used to generate gene expression and TCR clonality signatures at the single cell level. The WES revealed a higher frequency of mutations in R/R T-PLL compared to their baseline counterparts. R/R T-PLL samples displayed mutations of CXCR4 (37%; p.S312fs), STAT5B (46%; p.N642H), ATM (82%; p.L2890V), SAMHD1 (92%; p.A565T) and BCOR (p.N1585fs) and deletions in ASXL2 which were absent in treatment naïve T-PLL. In addition, all T-PLL samples harbored recurrent mutations on CXCR4, CD27, JAK1, STAT3, CCND1, CDKN1B, and the epigenetic regulators KMT2C, KMT2B, TET2, and KDM5C. The mutational profiles revealed the genomic complexity of T-PLL. The ubiquitous presence of activating mutations affecting the JAK/STAT axis underscored their importance in driving T-PLL survival. We also studied the diversity of TCR clonality and its relationship with pathogenesis and prognosis in T-PLL. All the samples exhibited dominance of at least one to up to 3 specific CDR3 sequences, revealing the anticipated clonal expansion in T-PLL. Expression of TCRα and TCRβ genes in the dominant clone differed among the samples.Monoclonality was validated as biallelic only when the 2nd largest transcripts of CDR3α or CDR3β clones were at least 5 times larger than its subceeder. The coupled TCR seq and RNA seq analysis displayed varied degrees of clonal expansions at a single cell resolution across all the patient samples. The T cell subsets were characterized by the expression of various T cell-markers across the cell clusters. In particular, 60% of the samples showed more expression of IL7R and lesser expression CD8A and CD8B, indicating their ability in suppressing the antitumor immunity. CD3E and CD3D were highly expressed across the clusters of T-PLL datasets.Out of 11 datasets, NKG7 and GZMA (cytotoxic T cell markers) were identified in more than 40% of the samples. The augmented levels of anti-apoptosis (BCL-2) and Jak/Stat signaling (JAK1, STAT3) markers were seen across the clusters of T-PLL scRNA seq datasets. The higher expression of PPP1R14B was found in 70% of the samples indicating its probable role in poor prognosis in T-PLL. In conclusion, T-PLL is characterized by numerous genomic aberrations, presence of 1-3 dominant clones, and gene expression signatures that facilitate survival, and resistance to apoptosis.

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with therapies targeting Bruton's tyrosine kinase (BTK) and BCL-2, but are limited due to the development of resistance. Dual targeting of BTK and BCL-2 has been efficacious in the clinic, with many patients achieving uMRD and prolonged remission off therapy. We have previously demonstrated the pre-clinical efficacy of LP-168 (Rocbrutinib), a novel selective 4th generation BTKi with an active warhead capable of covalent interaction with WT and T474I BTK and non-covalent binding when a BTK C481 mutation is present (Gordon et al. 2023). This allows for continued BTK inhibition despite development of common resistance mutations. To explore potential combination strategies with LP-168, we utilized a genome wide CRISPR/Cas9 knockout screen with validation of identified targets. Methods: Genome wide CRISPR/Cas9 knockout screening was performed in HG-3 CLL cells using the Brunello library with analysis via the MAGeCK pipeline. Cells were treated with IC20 (1 uM) of LP-168 for 3 days. Following exclusion of essential genes, negatively selected sgRNAs that were differentially expressed in the LP-168 group were used for analysis. Validation of hits was performed via pharmacological inhibition utilizing CellTiter-Glo, Annexin V/PI, and western blots using HG-3, OSU-CLL, TMD8 WT BTK, TMD8 C481S BTK, and TMD8 T474I BTK cells in addition to primary patient samples. Experiments using TMD8 cells were performed following CRISPR modification to insert either C481S or T474I BTK. Results: The CRISPR/Cas9 screen revealed 1875 genes with at least 3 out of the 4 sgRNAs depleted in both replicates of the LP-168 treated group vs control. KEGG/GO analysis demonstrated significant enrichment of regulation of mitochondrial membrane potential (p=0.00195) and reactive oxygen species pathways (p=0.00195). As BCL-2 interacts with both pathways, and was amongst the top-ranking genes depleted, we chose to target it with either venetoclax (BCL-2i) or with LP-118, a dual BCL-2/-xL inhibitor currently under investigation for R/R hematological malignancies. Pharmacologic inhibition of BTK + BCL-2, with either venetoclax (ven) or LP-118, showed synergistic reductions in proliferation of HG-3 (p<0.05, n=3; p<0.05, n=3, respectively), OSU-CLL (p<0.001, n=3, p<0.0001, n=3, respectively), and TMD8 WT BTK (p<0.05, n=3; p<0.01, n=3, respectively) cells at physiologically achievable concentrations (10 nM - 1 uM LP-168 and 10 pM - 10 nM ven or LP-118). LP-168 + ven or LP-118 induced significant cytotoxic effects in OSU-CLL (11%, p=0.001, n=3; 21%, p<0.00001, n=3, respectively) and TMD8 WT BTK (36.4%, p<0.00001, n=3; 66.7%, p<0.00001, n=3, respectively) cells following 72-hour drugging. Next, we utilized primary CLL B cells from treatment-naïve patients and found after both a 24- or 48-hour exposure, the combination of 1 uM LP-168 + 4 nM LP-118 (88%, p<0.00001, n=8) or 1 uM LP-168 + 4 nM ven (73%, p<0.00001, n=8) was able to induce cytotoxicity. Following a 2-hour drugging, LP-168 inhibited both BCR and AKT/mTOR signaling in primary CLL B-cells shown by reduced phosphorylation of BTK, ERK, AKT, and GSK3β, with both combination treatments inducing further inhibition of these targets (98%, p<0.0001, n=4; 49%, p<0.05, n=4; 37%, p<0.05, n=4; 46%, p<0.01, n=4, respectively). Further, we observed retained synergistic effect with both covalent (C481S; p<0.01, n=3) and non-covalent (T474I; p<0.001, n=3) BTKi resistance mutations at physiologically relevant concentrations. Finally, to determine if these combinations retain efficacy in both BTKi and venetoclax resistant settings of CLL, we utilized patient samples who were resistant to both BTKi and ven and treated them with combination of LP-168 + ven and found both combinations retained cytotoxic effect following 24- or 48-hour exposure. Conclusions : These data show combined use of LP-168 with pharmacological inhibitors targeting BCL-2 and BCL-xL display synergistic activity in CLL, even in the presence of mutations that mediate resistance to BTKi and BCL-2i. These data are consistent with clinical data showing dual targeting of BTK, with previous generation inhibitors, and BCL-2 has been effective in the clinic, with many patients achieving uMRD and prolonged remission off therapy and supports continued preclinical and future clinical investigation of LP-168 with inhibitors of BCL-2 and BCL-2/-xL.

T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive malignancy. While most cases of T-ALL occur in children, most deaths (4 out of 5) occur in adults. Though the exact reason for this disparity is not known, it could be related to limited response to chemotherapy seen in adults, as about half of patients in remission will relapse due to the development of resistance, highlighting the need for novel therapeutics. B-cell lymphoma-2 (BCL-2) family proteins are essential regulators of apoptosis. These proteins have either pro- (BAX, BAK) or anti- (BCL-2, BCL-xL, MCL-1) apoptotic effects and are targets in treating many hematological malignancies. BCL-2 is an anti-apoptotic protein that is overexpressed in ALL. Currently, the BCL-2 inhibitor, venetoclax, is being tested in combination with many other drugs in clinical trials. Nicotinamide phosphoribosyl-transferase (NAMPT) is a rate-limiting enzyme of the NAD+ salvage pathway and is upregulated in many cancers, including T-ALL. NAMPT works by combining nicotinamide and 5-phosphoribosyl-1-pyrophosphate into nicotinamide mononucleotide which is then used to create NAD+, restoring its levels. NAD+/NADH is a coenzyme that plays a significant role in many cellular processes, such as metabolism and DNA maintenance. While previous NAMPT inhibitors (NAMPTi) had significant anti-cancer effects, they also had major on-target toxicities. This is because complete inhibition of NAMPT disrupts critical biological functions in both normal and cancer cells. RPT-1G is a 1st-in-class small molecule hyperbolic inhibitor of NAMPT. This novel inhibitor reduces NAMPT enzymatic activity but never completely turns off the enzyme, allowing healthy cells to survive while severely affecting the cancer cells that are addicted to NAD+ for survival. Because of this, we hypothesize that RPT-1G can synergize with venetoclax leading to an increase of efficacy at lower concentrations compared to monotherapy with either drug. We first characterized BCL-2 dependencies in six T-ALL cell lines. Of these six, three were found to have minimal response to venetoclax when given a range of concentrations for 24 hours. When these same cells were tested with RPT-1G, a similar trend was observed with the venetoclax-insensitive cells. The venetoclax-insensitive cell lines required higher concentrations of RPT-1G to show any response when treated for 24 hours compared to the venetoclax-sensitive cell lines. However, when combined, these two drugs were able to greatly decrease viability compared to monotherapy with either venetoclax or RPT-1G at 24 hours. In summary, our study defines the efficacy of venetoclax and the novel hyperbolic NAMPTi, RPT-1G, alone and in combination in several T-ALL cell lines. These results suggest that the combination of these two drugs could perform better together than alone and holds significant future implications for patients with venetoclax-resistant T-ALL. Future studies will focus on testing RPT-1G alone and in combination with venetoclax and other FDA-approved therapies on PDX models and primary samples to further explore the utility of RPT-1G in treating T-ALL. Additionally, we aim to characterize the effects of RPT-1G on important biological processes in T-ALL that rely on NAD+. Overall, our findings contribute to a deeper understanding of how venetoclax-insensitivity could be overcome, paving the way for new treatment strategies in T-ALL.

Chronic Lymphocytic Leukemia (CLL) is the most common hematological malignancy in the United States, accounting for approximately 25% to 35% of all leukemias. Targeted therapies such as Bruton's Tyrosine Kinase (BTK) and B-cell lymphoma-2 (BCL-2) inhibitors ibrutinib and venetoclax have produced durable responses in CLL however, a subset of patients experience relapse and poor outcomes. This group with a poor prognosis is in urgent need of therapies that address the unique biology of treatment resistant CLL. To investigate mutant TP53's contribution to disease progression, we collected samples at baseline from patients entering ibrutinib treatment for front-line and relapsed/refractory CLL. Large-scale exome sequencing was performed on 270 patient samples revealing that 106 patients harbored some TP53 aberration, mostly in the DNA binding domain (DBD). Mutational analysis revealed missense mutations R175H and R248Q were among the most frequently occurring. These variants are known gain of function (GOF) mutations in solid tumors contributing to tumor progression and therapeutic resistance. Eighty-two percent of TP53 mutations in this cohort had a simultaneous deletion of the short arm of chromosome 17 (del17p) shown by fluorescence in situ hybridization (FISH). Del17p serves as an independent predictor of progression on ibrutinib as these patients had an inferior response to ibrutinib. These data lead us to hypothesize that the presence of TP53 mutations in CLL supports a shortened time to initial therapy and progression and relapse compared to patients without mutations in TP53. Wild type (WT) p53 is an essential transcription factor functioning as a tumor suppressor responsible for DNA damage repair, regulating apoptosis, and maintaining genomic stability. To describe mutant p53 activity, DNA damage was induced by ionizing radiation (IR) administered at 3Gy for 1 hour. WT but not mutant TP53 samples responded to DNA damage by stabilizing p53 protein expression. After stabilization, WT p53 strongly induced p21 protein, whereas p53 mutant expressing patient samples did not induce p21 protein in response to IR indicating a disrupted DNA repair pathway ex vivo. However, these studies did not show which genes are being targeted transcriptionally by mutant p53 nor how mutant p53 binds to and regulates these targets allowing these affected cells to subvert apoptosis. To further investigate this, we independently modeled this system in two cell lines. The HG3 and OSU-CLL lines are patient-derived lines created using CRISPR/Cas9 gene editing to introduce specific mutations at R175H and R248Q into WT p53 CLL. Homozygous knock-in of R175H and R248Q mutations on p53 was confirmed through Sanger sequencing on expanded single-cell clones. A profile of differentially expressed genes was revealed during RNA-seq analysis of mutant TP53 and WT TP53 cell lines. Genes overexpressed in relation to WT include transcription factors such as AFF3 and BCL11b and known tumor suppressors like KLHL14. Interestingly, a strong downregulation was seen in CYP4F3 across mutants whose upregulation is shown to be associated with resistance to chemotherapeutic agents such as fludarabine and targeted agents such as ibrutinib in the mutant TP53 setting. To determine whether p53 mutants in CLL exhibit a unique transcriptional signature like what is seen in solid tumors, we are conducting chromatin immunoprecipitation (ChIP) for p53 in these cell lines. In summary, our study reveals that TP53 mutations take advantage of mechanisms of resistance towards apoptosis-inducing therapies and impair the normal tumor-suppressive function of WT p53 which remains to be addressed in the clinic. These significantly worsen patient prognosis and accelerate relapse. Ongoing analysis will identify and characterize the action of these differentially expressed targets in CLL and highlight their role in facilitating survival in TP53 mutant CLL cells during therapy.

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

The apoptosis blockade in chronic lymphocytic leukemia (CLL) can be overcome clinically through inhibition of the anti-apoptotic protein BCL2 with venetoclax. However, emergence of venetoclax resistance is driven by heterogenous mechanisms. Among these, BCL2 mutations decrease the binding affinity of venetoclax; BCL2 hyperphosphorylation increases the anti-apoptotic function of BCL2 by strengthening its interactions with pro-apoptotic molecules; and upregulation of alternative anti-apoptotic proteins like BCL-XL and MCL1 cannot be overcome by venetoclax. Novel strategies to target venetoclax resistance effectively and safely are warranted to improve the outcomes of patients with CLL. We studied whether the protein degrader WH25244 is effective on mutant BCL2, hyperphosphorylated BCL2 and overexpressed BCL-XL in CLL cells, to overcome venetoclax resistance. WH25244 is a bifunctional molecule derived from navitoclax that recruits VHL E3 ligases to BCL2 and BCL-XL proteins, resulting in degradation via the ubiquitin-proteasome system. The requirement of VHL for its activity allows WH25244 to target BCL2 / BCL-XL dependent cancer cells while sparing platelets from toxicity (BCL-XL dependent but VHL low). Previously, we showed that OSU-CLL cells expressing BCL2 mutant proteins (G101V, F104L, R107_110dup or A113G CRISPR knock-in) were resistant to venetoclax, yet sensitive to WH25244 (EC50s ≤200 nM, 72h) due to degradation of BCL-XL and partial degradation of mutant BCL2 (2023 ASH abstract #4195). Here, we performed BH3 profiling to determine whether BCL2 mutant cells differ in their survival dependencies. G101V and F104L mutant OSU-CLL showed reduced sensitivity to the pan-apoptosis activator BIM, suggesting reduced apoptotic priming. F104L mutant cells additionally showed reduced sensitivity to BAD, suggesting reduced dependency on BCL2. Sensitivity to BCL-XL inhibition by XXA1_Y4eK peptide was not affected by the presence of BCL2 mutations. We studied the cellular levels of serine 70 (S70) phosphorylated BCL2 (pBCL2) upon treatment with WH25244, its negative control (WH25244-NC) lacking an active VHL ligand, navitoclax, and venetoclax. As observed by western blot, WH25244 and venetoclax similarly reduced pBCL2 levels in OCI-Ly1 cells. Venetoclax was ineffective at dephosphorylating BCL2 in OCI-Ly1 cells induced for venetoclax resistance, yet WH25244 retained activity. To determine whether WH25244 degrades BCL2 based on S70 phosphorylation status, we stably overexpressed phosphorylatable WT BCL2, or a non-phosphorylatable mutant (S70A BCL2) in Jurkat cells. WH25244 reduced S70 pBCL2 in WT cells, and degraded BCL2 in the non-phosphorylatable mutant cells, suggesting broad targeting of BCL2. 10 nM WH25244 effectively killed both cell lines, with 50% Annexin V positive cells observed at 24 hours. In primary CLL cells, we validated that treatment with WH25244 causes loss of S70 pBCL2 and apoptosis, in a VHL-dependent manner. We studied whether WH25244 can overcome venetoclax resistance driven by survival dependence on BCL-XL. As assessed by CellTiter-Glo at 24h, WH25244 was potent against four cell lines sensitive to the BCL-XL inhibitor A-1331852 (EC50s <50 nM), but resistant to venetoclax (EC50s> 5000 nM): MOLT-4 (EC50 = 4 nM), PF-382 (EC50 = 20 nM), SUP-T11 (EC50 = 30 nM), and CCRF-CEM (EC50 = 350 nM). Moreover, BCL2 was knocked out (KO) from the OSU-CLL cell line, which originally expressed both BCL2 and BCL-XL. BCL2 KO cells became resistant to venetoclax (plateau at 50% death) whereas WH25244 caused dose-dependent killing (EC50 = 25 nM, 72h) of the BCL2 KO cells. Last, we studied the effect of WH25244 against normal lymphocyte subsets from healthy donors. Apoptosis was detected at 18h of treatment by Annexin V / TMRM staining of CD19+ B cells, CD3+ T cells, and CD56+ NK cells via flow cytometry. Like venetoclax, WH25244 induced apoptosis of normal B cells in the nanomolar range. Toxicity to T cells remained low, and 100 nM WH25244 caused 1.7-fold more toxicity to NK cells than venetoclax. In conclusion, our results indicate that WH25244 can degrade mutant BCL2, hyperphosphorylated BCL2, and BCL-XL, overcoming multiple mechanisms that drive resistance to venetoclax. These data justify continued preclinical and future clinical investigation of WH25244 in CLL.